Department of Pharmaceutics,School of Pharmacy,Shenyang Pharmaceutical University,No.103 Wenhua Road, Shenyang 110016,PR China

Original Research Paper

Thin-f i lm hydration preparation method and stability test of DOX-loaded disulf i de-linked polyethylene glycol 5000-lysine-di-tocopherol succinate nanomicelles

Xiaoyu Ai,Lu Zhong,Handong Niu,Zhonggui He＊

Department of Pharmaceutics,School of Pharmacy,Shenyang Pharmaceutical University,No.103 Wenhua Road, Shenyang 110016,PR China

A R T I C L E I N F O

Article history:

Received 20 March 2014

Received in revised form

23 June 2014

Accepted 26 June 2014

Available online 3 July 2014

Nanomicelle

A novel redox-responsive PEG-sheddable copolymer of disulf i de-linked polyethylene glycol 5000-lysine-di-tocopherol succinate(P5kSSLV)was designed and synthesized.Thin-f i lm hydration method was used to prepare DOX-loaded P5kSSLV nanomicelle.To optimize the preparation technology,we investigate the effects of dosage,type of organic solvent, hydration temperature and time,and cryoprotectant on drug-loading content,encapsulation eff i ciency,particle size,and zeta potential.The mean particle size and zeta potential were determined by Zetasizer.The morphology of the P5kSSLV-DOX nanomicelles was visualized by transmission electron microscopy.The drug-loading content and encapsulation eff i ciency of P5kSSLV-DOX nanomicelle were investigated by UV.The drug-loading content,encapsulation eff i ciency,particle size,and zeta potential of the f i nal optimized nanomicelles were 4.58%,97.20%,30.21 nm and-0.84 mV,respectively.In addition,the stability of nanomicelles was investigated,which included dilution stability and storage stability.The results showed that P5kSSLV-DOX nanomicelle had good dilution stability and storage stability at 4°C.The preparation method of P5kSSLV-DOX nanomicelle with thinf i lm hydration method was practical and simple,which was valuable to be further studied.

? 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

1. Introduction

Many anti-tumor drugs(e.g.paclitaxel,doxorubicin)have poor solubility in water,which reduces the therapeutic effect and limits the clinical application.To solve the problem, many preparation techniques have been explored,such as emulsions,cyclodextrin inclusion compounds,nanosuspensions and so on[1].But the effect is unsatisfactory and the adverse drug reaction is still serious.Nanomicelle has become the most promising carrier due to its ability to improve drug solubility,prolong the circulation time and target tumor tissue[2].

Nanomicelle is an aggregate composed of amphiphilic dior tri-block copolymers,which aggregate into a core-shell structure by hydrogen bond,electrostatic interaction and van der Waals forces among the molecules.Compared with other drug delivery systems,nanomicelle has some advantageous characteristics:(i)The critical micelle concentration of nanomicelle is low,which has better thermodynamics and kinetics stability,and better dilution stability[3].(ii) Nanomicelle has a core-shell structure:the hydrophilic shell can prevent the absorption with plasma opsonic,and hydrophobic core can load the insoluble drug.(iii)Nanomicelle has a small size and narrow particle size distribution,which can accumulatein tumorviaenhanced permeability and retention(EPR)effect,therefore,has a better tumor-targeting ability[2,4].(iv)The structure of polymer can be modif i ed with ligands or targets,which can improve the active targeting property[5-7].(v)The preparation is simple,and the nanomicelle is easy to store. Therefore,nanomicelle has a better prospect in the treatment of cancer.

The preparation of nanomicelle can be classif i ed into two categories:chemicaland physicalmethods.Chemical method involves the covalent coupling of the drug to the polymer,after which the drug was encapsulated into the core by self-assembly.The preparation process contains a series of chemical reaction,which can be challenging and complicated.Thus,most of nanomicelles are prepared byphysicalmethod,whichincludesdirectdissolution method,dialysis method,self-assembly solvent evaporation method and thin-f i lm hydration method.The thin-f i lm hydration method is most often used due to its simplicity and practicability,and its ability to yield small and uniform particles.

In this study,a novel redox-responsive copolymer of disulf i de-linked polyethylene glycol 5000-lysine-di-tocopherol succinate(P5kSSLV)is designed and synthesized.With doxorubicin(DOX)as a model drug,DOX-loaded P5kSSLV (P5kSSLV-DOX)nanomicelle was prepared using thin-f i lm hydration method.To achieve the optimal prescription,we investigated the effects of dosage,type of organic solvent, hydration temperature,hydration time,and cryoprotectant on drug-loading content,encapsulation eff i ciency,particle size,and zeta potential.In addition,we also studied the stability of the prepared nanomicelle for a month.

2. Materials and methods

2.1. Materials

Doxorubicin hydrochloride(DOX·HCl)was purchased from Beijing Huafeng United Technology Co.,Ltd.Triethylamine, dichloromethane,chloroform,methanol et al.were obtained from Yuwang Co.Ltd(Shandong,China).N-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP)and amidoethyoxyl-lysinedi-tocopherol succinate(amidoethyoxyl-LV)were synthesized in this laboratory.Methoxy PEG Thiol(mPEG5000-SH,MW 5000) was obtained from Beijing JENKEM TECHNOLOGY(Beijing, China).

2.2. Synthesis of P5kSSLV copolymer

Amidoethyoxyl-LV was dissolved in dichloromethane and reacted with SPDP to obtained PDP-LV,Then PDP-LV was reacted with mPEG5000-SH in dichloromethane to obtained the end-product P5kSSLV.

2.3. Preparation of P5kSSLV-DOX nanomicelles

20 mg P5kSSLV copolymer,triethylamine and DOX·HCl solution(1 mg/ml in methanol)were dissolved in organic solvent and were mixed for 1 h.Then the organic solvent was removed using a rotary vacuum evaporation and was further dried under a constant f l ow of nitrogen for 30 min to form a drug-containing lipid membrane.1 mL HEPES buffer solution(HBS,pH 7.4)was added to the drug-containing lipid membrane and the solution was heated and stirred to get a preliminary nanomicelle solution.Such nanomicelle solution was then centrifuged at 13,000 rpm for 20 min,and the f i nal nanomicelle was obtained by f i ltering through 0.22 μm f i lters.

2.4. Optimization of thin-f i lm hydration method

2.4.1. Optimization of organic solvent

Nanomicelle was prepared as the method described in 2.2. Theorganicsolventwasdichloromethaneortrichloromethane.Then the drug-loading content,encapsulation eff iciency and particle size were measured as described.

2.4.2. Optimization of hydration temperature and time

Nanomicelle was prepared as the method described in section 2.2.The hydration temperature was 25°C,37°C,50°C,60°C and the hydration timewas 3 h,4 h,5 h,6 h,respectively.Then P5kSSLV-DOX nanomicelles were evaluated.

2.4.3. Optimization of proportion of DOX and P5kSSLV

Nanomicelle was prepared as the method described in 2.2. The amount of DOX was 0.5 mg,1 mg,2 mg and 3 mg, respectively. Then P5kSSLV-DOX nanomicelles were evaluated.

Fig.1-Synthetic route of copolymer P5kSSLV.

2.4.4. Optimization of lyoprotectant

The nanomicelle solution was added 5%or 10%of sucrose or mannitol.Then the particle sizes of nanomicelle solution were measured before and after freeze-drying.

2.5. Drug-loading content(DL)and encapsulation eff i ciency(EE)

Ultraviolet-visible light spectrometer was used to measurethe DL and EE of P5kSSLV-DOX nanomicelles and the absorption wavelength was 485 nm.P5kSSLV-DOX nanomicelle solution was centrifuged for 20 min at 13000 rpm to remove the free DOX.Then P5kSSLV-DOX nanomicelle solution was diluted with methanol for 20-fold and was sonicated for 30 min to disassemble the nanomicelles,which facilitates the encapsulated DOX to dissolve.After f i ltration through a 0.22 μm f i lter,the optical absorbance of the solution was measured. The DOX content was calculated from the calibration curve, which had a linear range of 15-40 μg/ml.The drug-loading percentage was def i ned as the ratio between the amount of the DOX content and the amount of drug-loaded nanomicelles.The encapsulation eff i ciency was calculated as the percentage of the encapsulated DOX among the total amount of DOX.The formulas are as follow:

DL%=weight of the drug in nanomicelles/weight of the feeding copolymer and drug × 100

EE%=weight of the drug in nanomicelles/weight of the feeding drug × 100.

2.6. Particle size and zeta potential

The particle size and zeta potential of P5kSSLV-DOX nanomicelles were determined by Zetasizer(Nano ZS,Malvern Co., UK).The measurements were repeated three times.

2.7. Particle morphology

The morphology of the micelles was characterized using transmission electron microscope(TEM,H-600,Hitachi, Japan).Before visualization,diluted dispersions of micelles were negatively stained with 1%phosphotungstic acid.

2.8. Stability of nanomicelle

2.8.1. Dilution stability

The nanomicelle solution was diluted with phosphate buffer solution for 10,100,1000,2000times,respectively.The particle sizes were measured.

2.8.2. Storage stability

The nanomicelle solution was saved at 4°C.The drug-loading content,encapsulation eff i ciency,zeta potential and particle size were investigated at predetermined time intervals for a month.

3. Results and discussion

3.1. Synthesis of P5kSSLV copolymer

The hydrophilic group mPEG5000and hydrophobic group amidoethyoxyl-lysine-di-tocopherol succinate(amidoethyoxyl-LV)were linked by disulf i de bond crosslinking agent N-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP).SPDP reacted with amidoethyoxyl-LV f i rstly and reacted with mPEG5000-SH secondly.The synthesis route was shown in Fig.1.

3.2. Preparation of nanomicelles

Fig.2-Schematic of the self-assembly of P5kSSLV-DOX nanomicelles in aqueous medium and the process of thin-f i lm hydration method.

P5kSSLV copolymer and DOX were dissolved in organic solvent after a thorough mixing.Then the organic solvent was removed to get the drug-containing lipid membrane.Then such lipid membrane was dissolved in HBS for the selfassembly of P5kSSLV-DOX nanomicelles.The driving force of self-assembly was the hydrophobic effect between the hydrophobic segments in the polymer.The hydrophobic effect also played an important role in the encapsulation of DOX,which stabilized the intermolecular interaction between DOX and the hydrophobic segment.The PEG hydration shell shielded the DOX against the enzymatic degradation and phagocytosis in vivo,which would improve drug stability and therapeutic effect.Fig.2 showed the schematic of the selfassembly of P5kSSLV-DOX nanomicelles in aqueous medium and the process of thin-f i lm hydration method.

Table 1-Optimization of organic solvents.

Fig.3-Encapsulation eff i ciency of P5kSSLV-DOX nanomicelles in different hydration temperature and hydration time.

3.3. Optimization of the preparation conditions for thinfi lm hydration

3.3.1. Organic solvent

Fig.4-Drug-loading content of P5kSSLV-DOX nanomicelles in different hydration temperature and hydration time.

Table 2-Optimization of proportion of P5kSSLV and DOX.

Table 3-Optimization of cryoprotectant.

Organicsolventaffectsthepreparation ofnanomicelle through two aspects.One is the extent of mixing between drug and copolymer,the other is the extent of the extension of copolymer molecules.Usually,organic solvent in thin-f i lm hydration method is volatile,with chloroform being the most commonly used.However,due to the strong liver and renal toxicity of chloroform,we chose dichloromethane as the organic solvent to investigate the preparation effect in comparison with chloroform.Table 1 showed that there were nearly no differences between dichloromethane and chloroform in particle size,drug-loading content and encapsulation eff i ciency.For safety concerns,we chose dichloromethane as the optimal organic solvent.

3.3.2. Hydration temperature and hydration time

Fig.5-Transmission electron microscopy(TEM)image of P5kSSLV-DOX nanomicelle.

The hydration temperature in preparation method was investigated at 25°C,37°C,50°C,60°C and the hydration time was 3 h,4 h,5 h,6 h,respectively.Particle sizes of nanomicelles were similar under different hydration temperatures and time.Sowetookthedrug-loadingcontentand encapsulation eff i ciency as the target to optimize the preparation technology.As shown in Figs.3 and 4,the drug-loading contents and encapsulation eff i ciency were reduced as temperature increased.At 50°C and 60°C,the drug-loading contents were below 3.5%and encapsulation eff i ciencies were below 73%.This was probably caused by the degradation of DOX or P5kSSLV copolymer at higher temperature.Compared with 25°C,nanomicelles at 37°C had higher drug-loadingcontent and encapsulation eff i ciency.In addition,drugloading content and encapsulation eff i ciency increased with the extension of hydration time.After 5 h,drug-loading contentand encapsulationeff i ciencyremained relativelystable.It indicated that the DOX had come into the P5kSSLV nanomicelles as more as possible and the system had been stable. Therefore,37°C and 5 h were chosen as the optimal hydration temperature and hydration time.

Fig.6-The particle size changes of P5kSSLV-DOX nanomicelle with different dilution factor.

Fig.7-The particle size changes of P5kSSLV-DOX nanomicelle during 1 month storage at 4°C.

Fig.8-The zeta potential changes of P5kSSLV-DOX nanomicelle during 1 month storage at 4°C.

3.3.3. Weight ratio of P5kSSLV to DOX

The amount of P5kSSLV copolymer was f i xed at 20 mg,and different doses of DOX were added into the organic solvent to achieve different proportions between P5kSSLV and DOX (20:0.5,20:1,20:2,20:3,w/w).Table 2 showed the particle sizes of different proportions were similar,suggesting that the amount of DOX had minimal effect on the particle size.This might be due to the strong hydrophobic interaction between DOX and di-tocopherol succinate.All the zeta potential values werenearzero.ThiswasbecausethePEGchainformedneutral PEG hydration shell on the surface of nanomicelle,which would reduce the electronegativity of nanomicelles.The encapsulation eff i ciency of nanomicelle decreased as the dosageofDOXincreased.Theencapsulationeff i ciencyat20:0.5 (97.31 ± 1.15)wasclosetothatof20:1(97.20 ± 1.02),butthedrugloading content was much higher at 20:1.Therefore,20:1 was used as the optimum proportion between P5kSSLV and DOX.

Fig.9-The drug-loading content changes of P5kSSLV-DOX nanomicelle during 1 month storage at 4°C.

3.3.4. Cryoprotectant

For long-term storage,it is ideal for P5kSSLV-DOX nanomicelles to be freeze-dried.Particle size was measured after different cryoprotectants was used to ref l ect the quality of freeze-drying procedure.As shown in Table 3,no cryoprotectant and mannitol yielded much bigger particle size than sucrose.The relativelysmaller particlesize after freeze-drying with sucrose was much closer to the particle size before lyophilization,which ref l ected a better quality of cryoprotectant.Therefore,5%sucrose was chosen as the cryoprotectant. In summary,the optimal preparation method was determined as follows:P5kSSLV-DOX nanomicelle was prepared with thin-f i lm hydration method;dichloromethane was chosen as the organic solvent;the hydration temperature and time were 37°C and 5 h,respectively;the proportion of P5kSSLV and DOX was 20:1(w/w),with the cryoprotectant being 5%sucrose.

3.4. Particle morphology

The particle morphology of P5kSSLV-DOX nanomicelle was investigated with TEM.Fig.5 showed the nanomicelles were sphericalwith relativelyhomogeneoussizedistribution around 14-21 nm in diameter.

3.5. Stability study of nanomicelle

P5kSSLV has a low CMC value,which can self-assemble into nanomicelles easily and maintain the structural integrity.To examine the dilution stability,P5kSSLV-DOX nanomicelles solution was diluted with double distilled water,and the particle size was measured.As shown in Fig.6,the particle size was about 30 nm within 100 times dilution.Then the size increased to 78.21 nm at 1000 times dilution and 104.6 nm at2000 times dilution.The increased sizes suggested the nanomicelle was on the verge of disassemble,but still maintained the basic structure of micelle.The results indicated P5kSSLVDOX nanomicelles had high dilution stability.

Fig.10-The encapsulation eff i ciency changes of P5kSSLVDOX nanomicelle during 1 month storage at 4°C.

We further investigated the storage stability of P5kSSLVDOX nanomicelle during 1 month of storage at 4°C Figs.7-10 showed the particle size,zeta potential,drug-loading content and encapsulation eff i ciency had little change during the onemonth period,which suggested that P5kSSLV-DOX nanomicelle had a high storage stability at 4°C storage.This was due to hydrophilic PEG chain increasing the dispersity of the nanomicelles in water and preventing the aggregation between nanomicelles.

4. Conclusions

A redox-responsive P5kSSLV-DOX nanomicelle was prepared with thin-f i lm hydration method.The dosage of DOX,type of organic solvent,hydration temperature and time,and cryoprotectant were investigated to optimize the preparation method.The prepared nanomicelle has small particle size, uniform size distribution and high encapsulation eff i ciency of DOX.In addition,P5kSSLV-DOX nanomicelle has high dilution stability and storage stability.Taken together,thinf i lm hydration method was an ideal method to prepare P5kSSLV-DOX nanomicelle that could maintain a long-term stability.

R E F E R E N C E S

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*Corresponding author.Tel./fax:+86 24 23986321.

E-mail addresses:hezhonggui@vip.163.com,hezhgui_student@aliyun.com(Z.He).

Peer review under responsibility of Shenyang Pharmaceutical University

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http://dx.doi.org/10.1016/j.ajps.2014.06.006

1818-0876/? 2014 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

Doxorubicin

Thin-f i lm hydration method

Single factor