MCT mixed micelles as carriers of

of disulfiram for improving plasma stability and antitumor. 2 effect in vivo. 3. 4. Linlin Miaoa, Jia Sua, Xuezhi Zhuoa, Lifeng Luoa, Yihan Konga, Jin...
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mPEG5k-b-PLGA2k/PCL3.4k/MCT mixed micelles as carriers of disulfiram for improving plasma stability and antitumor effect in vivo Linlin Miao, Jia Su, Xuezhi Zhuo, Lifeng Luo, Yihan Kong, Jingxin Gou, Tian Yin, Yu Zhang, Haibing He, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01094 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Molecular Pharmaceutics

1

mPEG5k-b-PLGA2k/PCL3.4k/MCT mixed micelles as carriers

2

of disulfiram for improving plasma stability and antitumor

3

effect in vivo

4

5

Linlin Miaoa, Jia Sua, Xuezhi Zhuoa, Lifeng Luoa, Yihan Konga, Jingxin Goua, Tian

6

Yinb, Yu Zhanga, Haibing Hea, Xing Tanga,*

7

8

a

9

Road, Shenyang 110016, China

Department of pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, NO. 103 Wenhua

10

b

11

110016, China

School of Functional Food and Wine, Shenyang Pharmaceutical University, NO. 103 Wenhua Road, Shenyang

12

13

*

14

E-mail address: [email protected]

15

Telephone number: 86-24-23986343

16

Fax: 86-24-23911736

Corresponding author: Xing Tang

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Abstract:

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The clinical application of disulfiram (DSF) in cancer treatments is hindered by its

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rapid

20

poly(ethyleneglycol)-b-poly(lactide-co-glycolide)/poly(ε-caprolactone)

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(mPEG5k-b-PLGA2k/PCL3.4k) micelles were developed for encapsulation of DSF -by

22

using the emulsification-solvent diffusion method. Medium chain triglyceride (MCT)

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was incorporated into the mixed polymeric micelles to improve drug loading by

24

reducing the core crystallinity. Differential scanning calorimetry (DSC) results

25

implied that DSF is likely present in an amorphous form within the micelles, and is

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well dispersed. DSF is encapsulated within the core and the reservoir is stabilized by

27

the hydrophilic shell to prevent rapid diffusion of DSF from the core. The DSF mixed

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micelles (DSF-MMs) showed good drug loading (5.90%) and a well-controlled

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particle size (86.4±13.2 nm). The mixed micelles efficiently protected DSF from

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degradation in plasma, with 58% remaining after 48 h, while almost 90% of DSF was

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degraded after the same period for the DSF solution (DSF-sol) which was used as a

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control. The pharmacokinetics study showed that the maximum plasma concentration

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and bioavailability of DSF were improved by using the DSF-MMs (2 and 2.5 times of

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the DSF-sol). The TIRs (tumor inhibition rates) of 5-FU, DSF-sol and DSF-MMs

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were 63.46%, 19.57% and 69.98% respectively, implying that DSF-MMs slowed the

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growth of a H22 xenograft tumor model effectively.

degradation

in

the

blood

circulation.

In

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study,

methoxy

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Molecular Pharmaceutics

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Keywords: disulfiram; mPEG5k-b-PLGA2k/PCL3.4k; mixed micelles; core crystallinity; plasma

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stability

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1 Introduction

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Disulfiram (DSF) has been used to treat alcoholism since the 1940's.1 It was reported

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that DSF’s metabolite S-Methyl-N, N-diethylthiocarbamate (Me-DTC) sulfoxide is a

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potent, irreversible inhibitor of low Km mitochondrial aldehyde dehydrogenase

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(ALDH).2 In recent years, DSF and its metabolites have been demonstrated to possess

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strong antitumor activity towards various tumor types.3 The anticancer activity of

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DSF is copper (Cu)-dependent,4, 5 and the combination of DSF and Cu triggers the

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generation of reactive oxygen species (ROS) and inhibits the activity of

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proteasome-NF-kB.6, 7 It has been demonstrated that DSF/Cu induces cell death via

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the combination of two actions: (1) instant death caused by ROS produced by DSF/Cu

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and (2) delayed and stronger cytotoxicity by diethyldithiocarbamic acid (DDC)-Cu.8

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Furthermore, DSF can also increase the sensitivity of 5-FU and other anticancer drugs

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towards

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diethyldithiocarbamate-copper complex (CuET),11 metabolite of DSF contributes to

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the major anti-tumor effects. It could bind NPL4 and induce NPL4 aggregation,

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consequently inhibiting p97 pathway. However, there has been little progress with

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regards to in vivo antitumor studies of DSF, which is of particular importance for oral

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administration. This is because DSF is quickly degraded in the gastrointestinal system,

tumor

cells.9,

10

Recently,

it

has

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reported

that

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with complete degradation occurring within 30 min in simulated gastrointestinal fluid

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(SGF).12 It has been reported that DSF is more stable at pH 6-7, compared to its

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stability in extremely acidic and alkaline conditions.13 The half-life of naked DSF in

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the bloodstream is only 4 min, which severely impedes its applications in the clinical

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field.14 The important metabolites of DSF are shown in Fig. 1. In the blood, DSF

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rapidly degrades to diethyldithiocarbamic acid (DDC), which is also unstable, and is

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further degraded to diethylamine and carbon disulphide. DDC is also a substrate of

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phase Ⅱ metabolism, which involves formation of diethyldithiomethylcarbamate

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(Me-DDC).

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diethylthiomethylcarbamate (Me-DTC), which is further oxidized to its corresponding

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sulphoxide and sulphone metabolites.15 It is for this reason that there is such a

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discrepancy between the anticancer activity of DSF in vitro and in vivo. Hence, it is

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imperative to develop an efficient drug delivery system to transport DSF to tumor

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tissues and thereby increase its therapeutic efficacy.

Me-DDC

also

undergoes

oxidative

biotransformation

to

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Amphiphilic block copolymers, which can self-assemble into structures with

74

hydrophobic cores and hydrophilic shells, have been extensively studied as potential

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delivery vectors for poorly water-soluble drugs.16,

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encapsulate the hydrophobic drugs in the core and thus increase their circulation time

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in the body, facilitating favorable bioavailability and biodistribution. Generally,

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physical encapsulation or chemical binding to the block copolymer can be used to

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encapsulate active substances into the polymeric micelles.18,

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encapsulation process requires suitable functional groups between the drug and

17

Such polymeric micelles can

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The conjugated

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Molecular Pharmaceutics

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polymer, so it is a more complicated strategy. Whereas,the physical encapsulation

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method is more widely used, and it will be discussed here. However, it is not easy to

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achieve high drug loading and the micelle should be stable enough to deliver an

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efficient drug dose to the desired site. The amount of drug loading and final stability

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of polymeric micelles are mainly dependent on the solubility of the drug, type of

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polymers and the interaction between the drug and the hydrophobic core of the

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polymeric micelles.16 To further increase drug stability and drug loading efficiency of

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polymeric micelles, additional hydrophobic materials or physical blending of different

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polymers can be utilized to alter the properties of the hydrophobic core.20,

21

90

Krishnadas

of

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egg-phosphatidylcholine into a DSPE-PEG2K system, the mixed polymeric micelles

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exhibited a greater solubilization of drug in comparison to the DSPE-PEG2K single

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polymeric micelle.22 Jin X et al. concluded that juglone loaded Poloxamer

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188/phospholipid mixed micelles exhibited a low toxicity and improved cellular

95

uptake.21,

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encapsulate and deliver hydrophobic drugs to a specific site.

23

et

al.

reported

that

by

inserting

a

small

percentage

Therefore, mixed micelles offer a promising approach to effectively

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Song et al. formulated a mixed system of mPEG-PLGA and PCL using the

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nanoprecipitation method,24 which increased the encapsulation efficiency and the area

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under the cure (AUC) of DSF 13.5 fold, while the DSF concentration of the

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nanoparticles decreased rapidly in phosphate buffered saline (PBS) containing 10%

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fetal bovine serum (FBS) after 24 h. Duan et al. successfully entrapped DSF with a

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drug loading content over 5% by utilizing a shell cross-linked micelle system.25 5

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However, the materials used in this case were not degradable and the structure was

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complex, and thus the system was difficult to apply in the clinic. Despite these

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attempts, it still remains challenging to encapsulate DSF into amphiphilic

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nanoparticles,26, 27 and these limitations compromise the clinical applications of DSF.

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Medium chain triglyceride (MCT) is frequently used to prepare lipid-based

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formulations, such as emulsions, nanocapsules, and lipid nanoparticles. It is

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considered as a safe and biocompatible excipient, and shows good solubility for some

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extremely lipophilic drugs. Gou et al. demonstrated the feasibility of improving drug

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loading through physical blending of MCT into micelle cores.28 Biodegradable

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polymers such as (lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) have

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been extensively used to prepare nanocarriers for drug entrapment.29 Based on this,

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the aim of this work was to develop novel DSF-loaded mixed micelles (DSF-MMs) to

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increase drug stability and bioavailability. The micelles were prepared with

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PEG5k-b-PLGA2k/PCL3.4k and MCT by the emulsification-solvent diffusion method.

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To evaluate the potential of the drug delivery system, the optimal formulation was

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further characterized in terms of its in vitro physicochemical properties, including

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morphology, particle size, zeta potential, drug-loading capacity, and drug release. In

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vivo pharmacokinetics, an anti-tumor efficacy test and toxicity tests were also

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undertaken.

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2 materials and methods

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2.1Material 6

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Methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA; Mw:

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mPEG 5000 Da, PLGA 2000 Da, LA/GA75/25) and poly(ε-caprolactone) (PCL, Mw:

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3400 Da) were provided by Changchun Institute of Applied Chemistry (Changchun,

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China). DSF was obtained from the Department of Medicinal Chemistry, Shenyang

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Pharmaceutical University (Shenyang, China). Medium chain triglycerides (MCT)

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were purchased from Tieling Pharmaceutical Co. Ltd (Tieling, China). F68 was

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purchased from BASF AG (Ludwigshafen, Germany). Methanol and acetonitrile were

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obtained from Tianjin Concord Technology Co. Ltd (Tianjin, China). All other

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reagents were of analytical grade.

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2.2 Preparation of DSF-MMs

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DSF-loaded mixed micelles were prepared by the emulsification-solvent diffusion

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method using PCL3.4k and mPEG5k-b-PLGA2k as carrier materials. The optimized

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formulation and preparation process were as follows: PCL3.4k and mPEG5k-b-PLGA2k

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(5:5, w/w), 15% (of the material) MCT and 10% DSF were dissolved in ethyl acetate:

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benzyl alcohol (8:2) at 35 °C, and the obtained solution was used as the organic phase.

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0.3% poloxamer 188, 2% benzyl alcohol and 5% ethyl acetate were dissolved in water

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and heated to 35 °C. The organic phase was then slowly added to the water phase,

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followed by high speed shear mixing to obtain the O/W coarse emulsion. The coarse

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emulsion was subjected to high pressure homogenization at 600 bar for 4 cycles to

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yield the final emulsion. The same volume of cold water was added into the final

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emulsion with stirring under 10 °C, and the resulting colloid was purified and 7

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concentrated with tangential flow filtration by membrane module. Finally, the

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obtained DSF-MMs were passed through a 0.45 µm membrane syringe filter.

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2.3 Characterization of DSF-MMs

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The particle size and zeta potential of the obtained mixed micelles were determined

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by dynamic light scattering (DLS) using a NicompTM 380 Particle Sizing system (Zeta

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Potential/Particle Sizer NICOMPTM 380ZLS, Santa Barbara, California, USA).

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Morphological examination of the micelles was performed using a JEM-2100

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transmission electron microscope (JEOL, Japan) with an accelerating voltage of 200

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kV. The drug loading and encapsulation efficiency of DSF in micelles was determined

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by ultracentrifugation. The drug loading (DL%), drug encapsulation efficiencies

155

(EE%) and the recovery ratio (R%) were calculated by the following equations:

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DL (%) =

DSF weight ×100% total MMs weight

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EE (%) =

DSF content in micelles ×100% theoretical DSF content in micelles

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R (%) =

resulted DSF - MMs weight ×100% total material fed

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The solution was centrifuged at 3000 rpm for 15 min, and the filtrate was analyzed by

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HPLC on a Dima C18 column (4.6×200 mm, 5 µm) at 25 °C, using methanol and

161

water at a ratio of 8:2 (v/v) as the mobile phase. The elution was monitored at 254 nm

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at a flow rate of 1.0 mL/min. The thermal behavior of pure DSF, PCL3.4k,

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mPEG5k-b-PLGA2k, physical mixtures (PM) and DSF-MMs were evaluated by 8

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Molecular Pharmaceutics

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differential scanning calorimetry. Samples were accurately weighed (3-5 mg) and

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heated in closed aluminum pans at a heating rate of 10 °C/min from 20 to 100 °C

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under a nitrogen gas flow of 40 mL/min.

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2.4 In vitro release and plasma stability

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In vitro drug release was measured by a dialysis method. In brief, 1.0 mL DSF-MMs

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or DSF-sol (20 mg DSF was dissolved in Cremophor EL/ethanol of 1mL, and the

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obtained solution was diluted to 1 mg/mL with distilled water) was sealed in dialysis

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membranes (MWCO: 12 kDa), and then dispersed in 10 mL phosphate buffered

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solution (PBS) of pH 7.4 containing 2% w/v sodium dodecyl sulfate (SDS) with

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continuous shaking at 100 rpm. SDS was used to increase the solubility of DSF in the

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buffer solution. At pre-determined time intervals (0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72

175

h), all the release media was removed and replaced with an equal volume of fresh

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medium to continue the release process. The amount of released DSF was measured

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using the HPLC method after filtering through a 0.22 µm membrane filter.

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To determine the plasma stability of encapsulated DSF-MMs and DSF-sol, 0.1

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mL of each formulation was added into an eppendorf tube containing 0.9 mL blank

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plasma, with shaking at 37 °C. At set time intervals (0, 0.167, 0.5, 1, 2, 4, 6, 8, 12, 24

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and 48 h) 50 µL was removed and added to activated cartridges (with 1 mL methanol,

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1 mL ethylenediaminetetraacetic acid (EDTA, 0.05M). DSF was then eluted from the

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cartridges using 1 mL water (5% acetonitrile) and 1 mL acetonitrile and measured by

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HPLC. 9

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2.5 In vivo pharmacokinetic study and detection of DSF

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Rats were randomly divided into two groups (n = 3): (1) DSF-sol group and (2)

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DSF-MMs group. The formulations were injected at a single dose of 20 mg/kg into

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the tail vein. Blood samples (0.5 mL) were removed from the orbital venous plexus

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and collected into 1.5 mL EP tubes containing the same volume of stabilizer (0.9%

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sodium chloride, 0.64% sodium acetate and 0.8% Diethylenetriaminepentaacetic acid,

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pH 4.5) at 0, 15, 30, 45, 60, 120, 240 and 360 min after dosing. The samples were

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immediately centrifuged at 6,000 rpm for 5 min and the supernates were collected.

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DSF was extracted by the solid phase extraction (200 µL sample with 20 µL internal

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standard diphenhydromine added) and the concentration was determined by the

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validated UPLC–MS/MS method.17

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Analytic separation was achieved using a Syncronis C18 column (50×2.1 mm,

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1.7 mm particle size, Thermo). The mobile phases A and B were acetonitrile and

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H2O/formic acid 99.9:0.1 (V/V) +1 mM Ammonium acetate. The gradient (0.2

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mL/min) was as follows: initially at 20% A and reaching 80% A linearly in 0.8 min,

200

followed by 1.5 min at 80% A, then reaching 20% A linearly in 0.2 min. Finally,

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equilibrating at 20% A for 0.5 min. The column temperature was held at 40 °C.

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Quantitative determination of DSF was performed using a Waters ACQUITYTM

203

XEVOTQ mass spectrometer. Data was acquired in the electrospray ionization (ESI)

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mode with positive ion detection and multiple reaction monitoring (MRM). A cone

205

voltage of 14 V and capillary voltage of 3.50 kV were used. The desolvation 10

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Molecular Pharmaceutics

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temperature was maintained at 400 °C and nitrogen was used as the desolvation gas

207

with a flow rate of 600 L/hr. The MS/MS transition of DSF was 296.98 →115.93 and

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that of diphenhydromine was 256.14 → 167.02.

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2.6 In vivo anti-tumor efficacy test and toxicity tests

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Kunming mice (18-22 g) were acquired from the animal center, Shenyang

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Pharmaceutical University. All animal experiments were conducted in accordance

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with the guidelines of the Laboratory Protocol of Animal Care and Use Committee,

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Shenyang Pharmaceutical University. H22 tumor cells were intraperitoneally injected

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into healthy Kunming mice (0.2 mL/mouse), and inoculation of ascites in the mice

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was observed. After the abdominal cavity could be seen to be filled with ascites, mice

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were sacrificed and the ascites were diluted 50 times with saline. The H22 xenograft

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tumor model was prepared by inoculating the right armpit with 0.2 mL diluted ascites

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after iodophor disinfection and 75% alcohol disinfection. After tumor reached to

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100-200 mm3, the mice were randomly divided into four groups according to the size

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of tumor volume and the principle of group consistency. Grouping and dosing

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regimens are shown in Table. 1. The oral dosing of copper gluconate was at 19.2

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mg/kg 5-6 hours before tail vein injection of the DSF preparation. The samples were

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injected into the tail vein of each mouse for 3 weeks (twice a week). Tumor volume

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and body weight were used to evaluate the treatment efficacy and safety, respectively.

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At various time intervals, the tumor volume was measured by a vernier caliper and

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calculated using the following equation: V=1/2ab2, where a and b are the major and 11

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minor axes of the tumors. During the course of the experiment, the mice weights were

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weighed daily. Animals were sacrificed 2 weeks after drug administration using

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diethyl ether anesthesia, and the final size and weight of tumors were analyzed.

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2.7 Statistical analysis

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SPSS17.0 statistical software was used for the Student's t test. Results are given as

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mean values ± S.D. P values of < 0.05 were considered significant.

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3 Results and discussion

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3.1 Preparation of DSF-MMs

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It has been reported that different structures of PEG-PLGA copolymers with different

236

properties can be used for different drug delivery systems, such as nanoparticles,

237

micelles and hydrogels. When the PEG (fEO) fraction is above 45%, micelles were

238

found

239

polyethyleneoxide-polybutadiene

240

polyethyleneoxide–polyethylethylene

241

mPEG5k-b-PLGA2k was used, and the emulsification-solvent diffusion method shown

242

in Fig. 2 was used to prepare the DSF-MMs. The organic solvents were removed

243

using tangential flow filtration, simultaneously concentrating the formulation.

to

more

easily

form,

shown

(PEOm-PBDn)

in

a

and

(PEOm-PEEn).30

study

of

polymer

hydrogenated

homolog

In

this

study,

244

As summarized in Table 2, all the micelles were in the nanoscale size range, and

245

their size varied from 60 to 200 nm depending on the formulation and preparation

246

process. The ratio of organic phase to water phase had no significant effect on the size 12

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Molecular Pharmaceutics

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and drug loading of the nanoparticles, while increasing the PCL loading significantly

248

increased the size of the nanoparticles and the drug loading. It has previously been

249

reported that the use of PCL is necessary for DSF encapsulation.31

250

It has also been reported that the addition of MCT to the nanoparticle system

251

could improve the drug loading and stability without affecting the drug

252

pharmacokinetics in vivo.28 As MCT can penetrate into the polymer core and disrupt

253

the arrangement of the inner core, the amorphous area is expanded, which increases

254

the drug loading of the nanoparticles. According to our previous work, the maximum

255

drug loading of DSF could be increased from 2.61±0.10% to 8.34±0.20% by

256

incorporating MCT into mPEG-PCL polymeric micelles.28 Furthermore, it is known

257

that the degradation of DSF is less than 1 % in MCT at 60 °C for 10 days,26 and so we

258

prepared micelles with different MCT contents to investigate the effect of MCT on

259

drug loading. There is a gradual increase in drug loading upon MCT incorporation.

260

However, no obvious increase occurred when the MCT amount was over 30%, and

261

the particle size increased sharply. Interestingly, the particle size first decreased as the

262

amount of MCT incorporated was increased, and then increased as addition continued.

263

Glavas et al. reported that the reduction could be caused by the decrease in core

264

crystallinity.32 Polycaprolactone (PCL) is semicrystalline, and its crystallinity may be

265

altered by MCT, after which the core can pack tightly in the non-crystalline regions,

266

which further influences the core size and the nanoparticle size. The increased particle

267

size is thus possibly due to MCT expanding the core volume. Based on the above

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results, 15% MCT of the polymer weight was added to the formulation to acquire the

269

optimum drug loading of 5.90% and the recovery ratio was 82.0%.

270

Based on this, in subsequent experiments the feed ratio of DSF, PCL3.4k,

271

mPEG5k-b-PLGA2k and MCT was set as 2:10:10:3 (w / w) to prepare the DSF-MMs.

272

3.2 Characterization of DSF-MMs

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The schematic illustration of the DSF-loaded mPEG5k-b-PLGA2k/PCL3.4k/MCT mixed

274

micelles is shown in Fig. 2. The surface morphology of the DSF-MMs was observed

275

and a representative TEM image is presented in Fig. 3. The TEM image confirmed

276

that the micelle formulations were spherical in shape. The particle diameter of the

277

micelles was measured to be 86.4±13.2 nm using DLS analysis, which was consistent

278

with the TEM image. Particles showed good uniformity, and no oil droplets could be

279

seen embedded in the micelle cores when observed in the TEM images, which is

280

expected as the content of MCT is only 15% of the used materials, and is thus not

281

high enough to form an oil core. Zeta potential is correlated to the charge on the

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surface of a nanoparticle and can reflect particle’s stability to some extent. When the

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surface charge is high, there is a strong electrostatic repulsion between the

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nanoparticles, which results in reduced aggregation.33,

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negatively charged with a zeta potential range of -15 mV to -30 mV. Furthermore,

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zeta potential is an important feature in relation to the interaction of nanoparticles

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with plasma proteins, which is known to be influenced by the surface charge of the

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nanoparticles. The DSC thermograms of DSF powder, PM, PCL3.4k, DSF-MMs and 14

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The nanoparticles were

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mPEG5k-b-PLGA2k are shown in Fig. 4. Pure DSF, PCL3.4k, and mPEG5k-b-PLGA2k

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have distinct and sharp endothermic peaks at 70.55 °C, 59.46 °C, and 56.45 °C,

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respectively. The physical mixture showed the 59.45 °C and 70.83 °C peaks. Even

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though the peaks of PCL3.4k and mPEG5k-b-PLGA2k overlapped, this has no effect on

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the DSF peak. The disappearance of the DSF peak of the DSF-MMs suggests that

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DSF may be present in an amorphous or molecular form. As well, there is only a

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small peak at 52 °C and the peak of PCL3.4k disappears, which may reflect that the

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state of PCL3.4k is amorphous in the formulation. As the free drug has been removed

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during the preparation process, the encapsulation efficiency is high, and is thus no

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longer considered as an assessment indicator during the prescription and process

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screening.

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3.3 In vitro release and plasma stability

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In vitro release and plasma stability tests were carried out to predict the stability of the

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micelles in vitro. SDS was added to the release medium to increase the solubility of

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DSF and protect DSF from degradation. The release profile of DSF from DSF-MMs

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and DSF-sol is presented in Fig. 5, where it can be seen that there was no obvious

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sustained-release behavior for the DSF-MMs compared to the DSF-sol. As well,

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approximately 80% of DSF was released within 24 h from DSF-sol. Zhang et al.

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reported that DSF could be released to 80% at 12 h.35 The difference may be caused

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by the addition of polyoxyethylene castor oil, which could form micelles,

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encapsulating DSF and slowing the release. The release curve of DSF-MMs was 15

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modeled and its release mechanism was subsequently analyzed. It can be seen from

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Table. 3 that it conforms to the Ritger-Peppas model,36-38 indicating that the release

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mechanism of DSF-MMs is diffusion and erosion. Diffusion and core degradation are

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the main release mechanisms of micelles, and physically entrapped drugs are typically

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released mainly through the diffusion effect, and the characteristics of drug release are

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also relevant to the degradation of the copolymers. The hydrophobic core was

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degraded through hydrolysis of the ester linkage of the polymer, leading to sustained

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release of DSF.

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DSF is very unstable in the blood, forming DDC after rapid degradation, which

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soon breaks down into diethylamine and carbon disulfide. Thus, ensuring the stability

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of DSF in the blood is an important prerequisite for its anti-tumor effect. The stability

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of DSF-MMs and DSF-sol in the blood was evaluated by monitoring the

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concentration of DSF in plasma. As shown in Fig. 5b, the DSF-sol group degraded by

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16.03% after 1 h, while no degradation was seen for DSF-MMs. DSF-MMs and

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DSF-sol degraded to about 42.57% and 85.57% respectively after 48 h, and the DSF

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concentration remained above 60% of the total concentration for more than 50 h,

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demonstrating that DSF-MMs are more stable in plasma. This is in part attributed to

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the core-shell structure of the polymeric micelles. The outer hydrophilic PEG brush

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can introduce a steric effect and avoid interaction of the polymeric micelles with

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biological components, such as proteins and cells, which may lead to early micellar

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elimination by the RES. Micelles disassembling into single chains under the diluting

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conditions of blood circulation is another obstacle against plasma stability and 16

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development of effective micellar drug carriers. It is possible to prevent dissociation

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of the micelles by increasing the hydrophobicity of the block copolymer.

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Song et al.

24

evaluated the stability of mPEG-PLGA/PCL Mixed Nanoparticles

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in PBS solution containing 10% FBS, and the results showed that the size of the

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nanoparticles remained mostly constant, with only a slightly decreased intensity after

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48 h. However, obvious DSF degradation occurred after 24 h, so it could be

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concluded that the initial release of DSF was mainly due to diffusion, which had no

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effect on the size of the particles. These results appear to be consistent with the results

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shown here. The schematic diagram of the dissociation of the micelles and the release

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of DSF is shown in Fig. 6. According to the release profile, there are two distinct

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release stages, characterized by the change in the slope of the curves. Burst release

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may be attributed to the diffusion of DSF absorbed to the hydrophobic core. After this,

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the system showed a transition to a more gradual release trend, which is related to the

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diffusion of the drug from the inner side and the dissociation of the polymeric

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micelles. Regarding the plasma stability, interactions between the drug and protein

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molecules in solution may be responsible for the rapid release of the drug from the

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micelles observed. After this, the slow release is mainly controlled by diffusion, with

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little influence of decomposition.

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3.4 In vivo pharmacokinetic study

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A study was undertaken to compare the pharmacokinetics of the DSF-MMs to the

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DSF-sol. The plasma concentration-time profiles of DSF after injection of DSF-MMs 17

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or DSF-sol at a dose of 20 mg/kg in rats are shown in Fig. 7, and relevant

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pharmacokinetic parameters are listed in Table. 4. The plasma concentration of DSF

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in the mixed micelles group was much higher than that of the control group. The AUC

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of DSF in the mixed micelles group was nearly 2.5 times of that in the solution group

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(12084.0 versus 4706.4 µg/L*h). In comparison with the solution group, the

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DSF-MMs could prolong the half-life of the drug in the plasma (t1/2) from 1.07 h to

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2.50 h. These results implied that the clearance of DSF in plasma could be reduced by

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the polymeric micelles, owing to the long-circulating effect of PEG, and the

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DSF-MMs were able to enhance the stability and availability of DSF in vivo. These

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results agree with the plasma stability profiles, and address a key barrier against the

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successful in vivo delivery of DSF. The DSF plasma concentration of DSF-MMs is

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almost twice that of DSF-sol at all the time points, which could be attributed to the

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protection of the core-shell structure of the micelles. However, it could be seen to

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drop rapidly 15 min post-injection, either owing to fast distribution, rapid elimination

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or micelle dissociation. The outcome is partly consistent with the plasma stability

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experiment, which showed a sharp decline of DSF concentration after 30 min. As well,

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the decrease trends of the two curves are nearly the same, possibly because Cremphor

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EL can affect the disposition and pharmacokinetics of various drugs by changing the

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unbound drug concentration through micellar encapsulation.8 There are few reports on

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the in vivo mechanisms of block copolymer metabolism in the literature, however it

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can be speculated that PEG-PLGA has the same protection effect on DSF. The

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difference between DSF-MMs and DSF-sol was in the stability and the protection of 18

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Molecular Pharmaceutics

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micelles.

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3.5 In vivo anti-tumor efficacy test and toxicity tests

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A tumor growth inhibition study in Kunming mice (using H22 hepatocarcinoma cells)

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was performed, using a 40 mg/kg DSF equivalent injection of DSF-MMs or DSF-sol

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into the tail vein of rats. 20 mg/kg of 5-FU or saline were chosen as the positive or

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negative controls. The results suggested that the TIRs of 5-FU, DSF-sol and

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DSF-MMs were 63.46%, 19.57% and 69.98% respectively, confirming that

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DSF-MMs could produce a more pronounced tumor suppressing effect than DSF-sol,

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with an effect comparable to that of 5-FU. After 17 days of injections, the tumors of

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each group were removed and photographed, and the tumor growth inhibition rate

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was calculated, and these results are shown in Fig. 8A-C. The final tumor volume of

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the DSF-MMs was remarkably lower (P