Preparation, characterization and in vitro release properties of … · 2013-06-19 · The...

Preparation, characterization and in vitro release propertiesof morphine-loaded PLLA-PEG-PLLA microparticles via solutionenhanced dispersion by supercritical fluids

Fu Chen • Guangfu Yin • Xiaoming Liao •

Yi Yang • Zhongbing Huang • Jianwen Gu •

Yadong Yao • Xianchun Chen • Hu Gao

Received: 7 August 2012 / Accepted: 4 April 2013

� Springer Science+Business Media New York 2013

Abstract Morphine-loaded poly(L-lactide)-poly(ethylene

glycol)-poly(L-lactide) (PLLA-PEG-PLLA) microparticles

were prepared using solution enhanced dispersion by

supercritical CO2 (SEDS). The effects of process variables

on the morphology, particles size, drug loading (DL),

encapsulation efficiency and release properties of the

microparticles were investigated. All particles showed

spherical or ellipsoidal shape with the mean diameter of

2.04–5.73 lm. The highest DL of 17.92 % was obtained

when the dosage ratio of morphine to PLLA-PEG-PLLA

reached 1:5, and the encapsulation efficiency can be as

high as 87.31 % under appropriate conditions. Morphine-

loaded PLLA-PEG-PLLA microparticles displayed short-

term release with burst release followed by sustained

release within days or long-term release lasted for weeks.

The degradation test of the particles showed that the deg-

radation rate of PLLA-PEG-PLLA microparticles was

faster than that of PLLA microparticles. The results col-

lectively suggest that PLLA-PEG-PLLA can be a promis-

ing candidate polymer for the controlled release system.

1 Introduction

In recent years, the study of drug-controlled release system

has attracted great attentions in pharmaceutical technology

and drug delivery design. The control release system could

overcome the drawbacks of conventional dosages such as a

short elimination half-life, systemic toxicities and frequent

dosing [1]. Microspheres-based drug release systems are

the preference of researchers because of their flexible

administrations such as intramuscular, intravenous, lung

inhaled, etc. Besides, microspheres can be manufactured

with polymer materials owing to their good biocompati-

bility, biodegradability, absorbility and non-toxicity of

degradation products [2].

The technique of supercritical fluid (SCF) is a promising

way to prepare drug-loaded microspheres, avoiding some

intense conditions employed by conventional methods

and residual of organic solvent. Supercritical CO2 (scCO2)

is the most widely used supercritical anti-solvent due

to its favorable critical conditions (Tc = 304.1 K, Pc =

7.38 MPa), non-toxicity, lower cost, no/low organic sol-

vent residual, environmently benign, etc. [3]. In the pre-

vious study, small molecular drugs have been successfully

encapsulated into poly (L-lactic acid) (PLLA) microparti-

cles using supercritical CO2 fluid technique (SEDS pro-

cess) [4, 5]. In the SEDS process, the solution containing

the solute and SCF is co-introduced into the precipitation

chamber through a specially designed coaxial nozzle. The

SCF acts as both anti-solvent for its chemical properties

and ‘‘spray enhancer’’ by mechanical effect. The speed

streams of SCF contribute to smaller primary droplets for

more sufficient contact between solution and anti-solvent,

also the high velocity turbulent SCF is in favor of mix

and mass transfer, both generating higher super-saturation

and prompter precipitation. As a result, smaller and

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10856-013-4926-1) contains supplementarymaterial, which is available to authorized users.

F. Chen � G. Yin � X. Liao (&) � Y. Yang � Z. Huang � Y. Yao �X. Chen � H. Gao

College of Materials Science and Engineering, Sichuan

University, No.24, South 1st Section, 1st Ring Road,

Chengdu 610065, Sichuan, People’s Republic of China

e-mail: [email protected]

J. Gu (&)

Chengdu Military General Hospital, Tianhui Town,

Chengdu, People’s Republic of China

e-mail: [email protected]

123

J Mater Sci: Mater Med

DOI 10.1007/s10856-013-4926-1

http://dx.doi.org/10.1007/s10856-013-4926-1

finely dispersed particles can be produced by this method

[6, 7].

As is well known, PLLA is one of the most widely used

biomedical polymers for drug delivery systems owing to its

biodegradability and biocompatibility as well as the status

of regulatory approval [8]. However, its application has

been limited because of its hydrophobicity, low degrada-

tion rate and acidity of degraded products [9]. A useful

strategy to improve the hydrophilicity of the polymer as

carriers for hydrophilic molecules such as polypeptides

and proteins was to introduce hydrophilic segments of

poly(ethylene glycol) (PEG) into PLLA chains for poly

(L-lactide)-poly(ethylene glycol)-poly(L-lactide) (PLLA-

PEG-PLLA) block copolymer [10]. PEG is a hydrophilic,

biodegradable and biocompatible polymer that is used in

the pharmaceutical area to improve the biocompatibility of

the blood contacting materials. Thus the hydrophilic

domains of PLLA-PEG-PLLA copolymers acting as

modifier of hydrophobic PLLA networks could increase

loading and encapsulation efficiency of drug and protein

[11]. The release rate of drugs, loaded into microspheres

made of PLLA and PEG block copolymers, is also more

rapid compared to the same drugs loaded into PLLA

microspheres [12]. Besides, it can also accelerate the

degradation rate and decrease the acidity of degraded

products of the polymer. Moreover, a viscous aqueous

based carrier prepared from biocompatible and biode-

gradable polymer that can prevent particle migration at the

site of administration would be ideal for in vivo adminis-

tration of PLLA microspheres because of the lipophilic

nature [13]. So PLLA-PEG di-, tri-, or multi-block

copolymers for drug delivery and tissue engineering

applications have been intensively investigated [14, 15].

Morphine, as the most remarkable analgesic component

isolated from the opium since 1806, has been commonly

used for the management of post-operative and moderate to

severe cancer pain [16]. However, there are some disad-

vantages involved in the conventional dosages, for exam-

ple, a short half-life (1.7–3 h), demanding repeated

administrations of the same dose necessary every 4 h,

which periodically leads to transient high plasma concen-

trations bringing about some unwanted side effects such as

respiratory depression [17]. As a result, controlled release

morphine formulations with less frequent dosing and

attenuation of morphine-related adverse effects have been a

highlight. A controlled release morphine suppository dosed

twice a day was reported, showing no discomfort and other

side-effects apart from a temporary sedation and fatigue

[18]. Morphine sulfate extended-release formulations are

recommended by some researchers [19, 20], which contain

both immediate-release and extended-release components.

The immediate-release component provides an initial rapid

release of morphine to relief the pain, and the extended-

release component sustains therapeutic concentrations with

minimal peak-to-trough fluctuation for a demandable time.

In recent years, a few literatures about controlled release

morphine formulations for oral or rectal administration

have been reported and applied to clinical [18, 21, 22].

Most of the formulations were simple physical mixture

tablets or suppositories composed of drug and polymer

matrix through the moulding. The SEDS method provides a

promising way to produce controlled release morphine for

parenteral administration.

The objective of this study was to prepare morphine-

loaded PLLA-PEG-PLLA microspheres by SEDS process

for controlled release applications. The effects of the pro-

cess conditions on the surface morphology, particle size,

drug loading (DL), encapsulation efficiency, in vitro drug

release properties as well as biodegradation morphology

were investigated.

2 Experimental

2.1 Materials

Batches of PLLA-PEG(MW2000)-PLLA triblock copoly-

mer with three PEG contents (1, 3, 5 %) was purchased

from Daigang Biological Science and Technology Co. Ltd.

(Jinan, Shandong). PLLA (Mw = 100 kDa) was supplied

by Institute of Medical Polymers of Shandong (Jinan,

China). CO2 with a purity of 99.9 % was supplied by

Tuozhan Gas Co. Ltd. (Chengdu, China). Morphine

hydrochloride injection (MF, 10 mg/mL) was supplied by

Shenyang No.1 Pharmaceutical Factory (Shenyang, China).

Dichloromethane (DCM), Methanol (MeOH) and all other

compounds were of analytical purity.

2.2 Preparation of morphine-loaded PLLA-PEG-PLLA

microparticles

The SEDS apparatus and schematics of operation proce-

dure in this study were described in detail in the previous

Table 1 Process and formulation parameters addressed in the single

factor experiment

Parameter Component Unit Applied levels

a b c d

X1 mMF/mPLLA-PEG-PLLA 1:5 1:7.5 1:10

X2 mPEG/mPLLA-PEG-PLLA % 0 1 3 5

X3 P MPa 8 10 12 14

X4 CMFe mg/mL 4 8 12

e Morphine dissolved in MeOH


123

literature [23]. In this study, morphine and PLLA-PEG-

PLLA was dissolved in MeOH and DCM, respectively,

then they were mixed completely. Prior to use, the mor-

phine hydrochloride injection was dried in the oven at

37 �C for 12 h to remove the water, then collect the mor-

phine hydrochloride powder for the following experiments.

In the running of an experiment, when the desired pressure

and temperature were stabilized, the mixture solution was

delivered into the high-pressure vessel with a volume of

500 mL through the internal capillary of a stainless steel

coaxial nozzle which was installed on the top of the high-

pressure vessel. A HPLC pump (P3000, Knauer, Germany)

was employed to provide driving force in this process.

Simultaneously, scCO2 was introduced to the high-pressure

vessel through the external channel of the nozzle using a

high-pressure metering pump (2J-X8/32, Hangzhou Zhiji-

ang Petrochemical Equipment Co. Ltd., China). The inner

diameters of the two-channel coaxial nozzle were 50 and

800 lm, respectively. When the solution was sprayed into

the high-pressure vessel which was full of scCO2, a rapid

mutual diffusion at the interface between the scCO2 and the

mixture solution occurred instantaneously, then the organic

solvent was extracted into the scCO2, resulting in super-

saturation of the polymer and morphine and the formation

of microspheres. The different precipitation dynamic of

polymer and morphine corresponded to efficient encapsu-

lation of morphine into polymer matrixes. When the

spraying was finished, fresh scCO2 was continuously

delivered for 30 min to remove the residual organic sol-

vents in the products. Pure DCM was also delivered for

30 min to prevent the non-return valve of the HPLC pump

from blocking. During the washing process, the tempera-

ture, pressure and flow rate of CO2 were maintained as

described above. After washing, the vessel was gradually

depressurized to atmospheric pressure to collect the prod-

ucts for further analysis.

In this study, the temperature, flow rate of CO2 and flow

rate of the solution were kept at 308 K, 300 and 0.5 mL/

min, respectively. The effects of the ratio of drug to

copolymer (X1), the content of PEG in the PLLA-PEG-

PLLA triblock copolymer (X2), the precipitation pressure

(X3) and the concentration of morphine in MeOH (X4)

were investigated. Each process parameter was investi-

gated at three or four levels listed in Table 1.

2.3 Characterization of microparticles

The surface morphology of the microparticles was

observed using scanning electron microscope (SEM,

S4800, Hitachi, Japan). Before observation, microparticles

were glued on a standard stand using a double-sided tape

and then coated with a thin layer of gold. The mean particle

size and particle size distribution of microparticles were

analyzed using a laser particle size analyzer (Rise-2008,

Shandong, China). Approximately 5 mg microparticles

were suspended in the sample cell filled with ethanol.

Before measurement, the suspension was dispersed by

ultrasonic waves with power 100 W for 1 min. Then the

circulating pump started at 1,250 rpm to circulate the

sample’s suspension. Analytic software was automatically

used to assay the mean particle size and particle size dis-

tribution. X-ray diffraction of the prepared microparticles

Table 2 Some results of samples prepared with different experiment parameters

Samples Experiment parameters Results

X1 X2 X3 X4 Davf (lm) TLg (%) ALh (%) EEi (%)

1 1:5 1 12 8 2.04 16.67 15.86 ± 0.79 51.84 ± 1.03

2 1:7.5 1 12 8 2.29 11.76 9.73 ± 0.40 69.01 ± 0.76

3 1:10 1 12 8 2.86 9.09 8.25 ± 0.25 79.01 ± 8.07

4 1:10 0 12 8 2.08 9.09 9.92 ± 0.16 56.64 ± 0.49

5 1:10 3 12 8 3.59 9.09 9.26 ± 0.40 87.31 ± 6.36

6 1:10 5 12 8 5.73 9.09 9.38 ± 0.31 67.39 ± 2.93

7 1:10 3 14 8 5.03 9.09 8.92 ± 0.98 63.12 ± 7.49

8 1:10 3 10 8 2.59 9.09 8.71 ± 0.49 61.48 ± 3.74

9 1:10 3 8 8 3.09 9.09 9.34 ± 0.21 61.38 ± 3.17

10 1:5 3 12 4 2.81 16.67 17.92 ± 0.19 69.57 ± 4.77

11 1:5 3 12 8 3.08 16.67 17.40 ± 0.93 71.06 ± 4.74

12 1:5 3 12 12 3.60 16.67 16.83 ± 0.22 74.76 ± 8.27

f Mean particle size measured using a laser particle size analyzerg Theoretical drug loading in microparticlesh Actual drug loading in microparticlesi Encapsulation efficiency of drug in microparticles


123

(XRD) was carried using a Philips X’Pert MDP diffrac-

tometer. The measurement was performed in the range of

0�–50� with a step size of 0.02� in 2h using Cu Ka radi-

ation as the source. Fourier transform Infrared (FTIR) was

performed using a NEXUS spectrometer 670 (Thermo

Nicolet, USA) in transmission mode with a wave number

range of 4,000–400 cm-1. Approximately 1 mg of micro-

particles was mixed with KBr and pressed into a thin tablet.1H-NMR spectrum was measured at 400 MHz with a

UNITY INOVA 400 (Bruker, German) using tetramethyl-

silane as the internal standard.

2.4 Determination of drug loading and encapsulation

efficiency

DL and encapsulation efficiency are important parameters

in the evaluation of the properties of drug-loaded micro-

particles. To determine the DL, accurately weighed 30 mg

drug-loaded microparticles was dissolved in 5 mL DCM

and then 40 mL phosphate buffered saline (PBS, pH 7.4)

was added and stirred in a water-bath at 30 �C using a

magnetic stirrer to volatilize the DCM. The resulting

solution was filtered through a 0.22 lm membrane to

remove the precipitated polymer and the amount of mor-

phine was analyzed using a UV spectrophotometer (U3010,

Hitachi, Japan) at 284 nm. For the measurement of

encapsulation efficiency, another 30 mg of drug-loaded

microparticles was suspended in 10 mL ethanol to wash off

the unencapsulated or adsorbed drug. The suspension was

then centrifuged using a high-speed centrifuge (TG-18,

Chengdu, China) at 12,000 rpm for 10 min. Afterwards,

the supernatant was discarded and the precipitation was

dissolved in 5 mL DCM. The following procedures were

the same as the DL procedures as described above. Each

experiment was performed in triplicate. The DL and

encapsulation efficiency were calculated using Eqs (1) and

(2), respectively.

Drug loading = W1=W2� 100% ð1ÞEncapsulation efficiency ¼W3=W1� 100 % ð2Þ

Where W1 is the actual loading of morphine in the mi-

croparticles; W2 is the total weight of the microparticles;

W3 is the weight of morphine in the microparticles after

washing off the unencapsulated morphine.

2.5 In vitro drug release

Accurately weighed 20 mg drug-loaded microparticles

were loaded into the pretreated dialysis bag (Mw = 8,000-

14,000, Long March of the Glass Co., Ltd., Chengdu,

China). Then the dialysis bag was immersed in a wide-

mouth bottle with 100 mL phosphate buffered solution

(PBS, pH 7.4) and incubated in a water-bath shaker at

37 �C with stirring. In preset time interval, 10 mL of

Fig. 1 SEM micrographs of

samples precipitated by SEDS

with the ratio of morphine to

PLLA-PEG-PLLA at 1:5

(a sample 1), 1:7.5 (b sample

2)) and 1:10 (c sample 3)


123

released solution was periodically removed and 10 mL of

fresh PBS was periodically added to continue release. The

morphine solution withdrawn from the wide-mouth bottle

was measured using UV Spectrophotometer (U3010, Hit-

achi, Japan) at 284 nm and the release profiles were plotted

in terms of cumulative release percentage of morphine

(wt%) with time. Each experiment was carried out in

triplicate.

2.6 In vitro degradation properties

Polymeric microparticles degradation must be taken into

account in the design of drug delivery systems. For each

polymer sample, wide-mouth bottle was used with 20 mg of

microparticles and 100 mL of phosphate buffer (pH 7.4).

They were incubated in the water-bath shaker at 37 �C with

stirring. At suitable intervals of time, the degradation

medium containing microparticles was removed, then cen-

trifuged and dried for analysis. The morphology changes of

the samples were analyzed by SEM (S4800, Hitachi, Japan).

3 Results and discussion

3.1 Effect of process parameters

The experimental conditions and some results are sum-

marized in Table 2. The results indicated that the process

parameters had important effect on particle size, DL,

encapsulation efficiency. Figure 1, 2, 3, and 4 showed the

SEM photographs of the drug-loaded microparticles pre-

pared with various experimental conditions. All the

microparticles were successfully precipitated in spherical

or ellipsoidal shape with smooth surface. Apart from well-

shaped morphologies and clinically acceptable particle size

for intravenous injection [24], the encapsulation efficiency

and release properties were also crucial characterizations

for screening optimal conditions of preparing particles. The

actual DL was slightly higher than the theoretical DL due

to some weight loss of PLLA-PEG-PLLA (partly dissolved

in CO2 at supercritical condition) during the SEDS [25].

3.1.1 Ratio of morphine to PLLA-PEG-PLLA effect

The effect of the ratio of morphine to PLLA-PEG-PLLA

on the precipitation of the drug and polymer by the SEDS

process was studied with other fixed experimental condi-

tions. The morphology of the microparticles was shown in

Fig. 1, without significant differences between each other,

which were all spherical or ellipsoidal shape with slight

agglomeration. But it is worth noting that these samples

were non-uniform, including two different sizes, one in the

nanometer range, the other ranging from submicometric to

micron scale. This can be explained by the particle for-

mation mechanisms during the SEDS [26]. In the process



with different PEG contents in

PLLA-PEG-PLLA at 0 % (w/w)

(a sample 4), 1 % (w/w)

(b sample 3), 3 % (w/w)

(c sample 5) and 5 % (w/w)

(d sample 6)


123

jet break-up time (T) and dynamic surface tension van-

ishing (t) were considered as mechanisms in competition.

When T \ t, jet break-up prevails (always at subcritical

conditions, sometimes also at supercritical conditions),

droplets were formed and their subsequent drying produced

the microparticles. When T [ t, gas mixing dominated and

the particles precipitate formed in the absence of surface

tension, resulting in irregularly spherical nanoparticles

[27]. In this study, the two processes both existed, thus

producing particles with coexistence of two different

morphologies. The particle size of the microparticles

increased from 2.04 to 2.86 lm which can be observed

from Table 2. It could be explained in terms of super-sat-

uration, which is the driving force of nucleation and growth

[28]. With higher super-saturation, the nucleation domi-

nates, resulting in smaller particles, otherwise, the growth

prevails and produces larger particles. As MeOH is a poor

solvent for PLLA-PEG-PLLA, the reduction of the volume

fraction of MeOH in mixed solvent with the decrease of

morphine ratio would reduce the super-saturation of PLLA-

PEG-PLLA and form larger particles. The actual DL

increased with the increase of theoretical dosage while the

encapsulation efficiency decreased. The precipitated mor-

phine particles might act as host particles, which lead to

easy encapsulation of morphine by the precipitation of

PLLA-PEG-PLLA particles. High encapsulation efficiency

might be attributed to the appropriate precipitation rate

between drug and polymer.

3.1.2 Content of PEG in PLLA-PEG-PLLA effect

The ‘soft’ segment PEG grafted on the PLLA-PEG-PLLA

made a great impact on the precipitation of microparticles.

The precipitated process was carried out by changing the

content of PEG in PLLA-PEG-PLLA from 0 to 5 % (w/w).

Higher content of PEG disabled the successful formation of

spherical or ellipsoidal shape. As shown in sample 3, 4, 5

and 6 of Table 2, the mean diameter increased from 2.08 to

5.73 lm with the PEG content in copolymer increased from

0 to 5 %, which can also be observed in Fig. 2. This can still

be explained in terms of nucleation and growth processes

[28]. In this study, the increase of PEG content decreased

the molecular weight of the PLLA-PEG-PLLA, conse-

quently increasing the solubility of copolymer in organic

solvent. Besides, the solubility of PEG in DCM was greater

than that of PLLA in DCM because of the close solubility

parameter. When sprayed into the scCO2, the super-satu-

ration of copolymer solution with higher PEG content was

delayed to reach, therefore, growth was the prevailing

mechanism and lager particles were formed. The encapsu-

lation efficiency of morphine in PLLA-PEG-PLLA also

increased from 56.64 to 87.31 % when PEG content ranged

from 0 to 3 %, which might attribute to the hydrophilicity of

PEG. As morphine is a kind of hydrochloride with excellent

hydrophilicity, the introduction of PEG could promote the

integration between the drug and copolymer [29]. However,

when the content of PEG reached 5 %, the encapsulation



at 8 MPa (a sample 9), 10 MPa

(b sample 8), 12 MPa (c sample

5) and 14 MPa (d sample 7)


123

efficiency showed a decline. This may owing to the delay of

super-saturation when PEG content was too high, so a lot of

morphine precipitated on the surface of particles [30],

leading to lower encapsulation efficiency. Anyway, the

encapsulation efficiency of microparticles can be improved

by the introduction of PEG and 3 % might be an appropriate

choice in this study.

3.1.3 Pressure effect

The mean diameter primarily decreased from 3.09 to

2.59 lm with the pressure varying from 8 to 10 MPa and

then increased to 5.03 lm when the pressure reached up to

14 MPa. The change was in agreement with the previous

study [31]. Increasing the pressure resulted in an increase

in the CO2 density, as a consequence the acceleration of the

mass transfer made the nucleation dominate and produced

smaller particles [32]. As the pressure continued to

increase, the diffusion coefficient of the scCO2 was

insensitive to the change and the density difference

between anti-solvent and solvent was narrowed, thus

reducing the driving force of mass transfer and prolonging

the super-saturation time. As a result, particles were not

precipitated individually, leading to lager particle size and

heavier agglomeration [33]. The DL of microparticles as

shown in Table 2 indicated that there was no significant

difference at different pressures.

The encapsulation efficiency ranged from 61.38 to

87.31 %. The highest encapsulation efficiency was

obtained at 12 MPa with the suitable diffusion coefficient

of scCO2.

3.1.4 Concentration of morphine in MeOH effect

The concentration of morphine dissolved in MeOH was

investigated in the range of 4–12 mg/mL with the fixed

ratio of morphine to PLLA-PEG-PLLA at 1:5. The volume

fraction of MeOH would decrease with the increase of

morphine concentration. As noted in Table 2, the particle

size increased in sample 10, 11 and 12, as well as a slight

increase of the encapsulation efficiency. This can also be

proved in sample 1, 2 and 3, with the ratio of morphine to

PLLA-PEG-PLLA varied from 1:5 to 1:10. So these can be

attributed to the combined influence of theoretical dosage

and MeOH volume fraction.

Figure 4 showed that the surface of microparticles was

rougher than those obtained above. More morphine pre-

cipitated on the surface or loosely bonded with micropar-

ticles owing to the increase of the morphine dosage.

To sum up, PEG content had the most important effect

on the properties of microparticles including morphologies,

particle size and encapsulation efficiency. Considering of

these characteristics comprehensively, the optimal param-

eters for preparing morphine-loaded microparticles were



with the concentration of

morphine dissolved in MeOH at

4 mg/mL (a-sample 10), 8 mg/

mL (b-sample 11) and 12 mg/

mL (c-sample 12)


123

with a ratio of morphine to PLLA-PEG-PLLA at 1:10, a

PEG content of 3 %, a pressure of 12 MPa and a morphine

concentration of 8 mg/mL.

3.2 FTIR characterization

FTIR spectra were performed to study whether the mor-

phine was encapsulated in the copolymer after SEDS.

Figure 5 showed the FTIR spectra in the region

500–4,000 cm-1 of morphine, PLLA-PEG-PLLA raw

materials, morphine and PLLA-PEG-PLLA physical mix-

ture and morphine-loaded PLLA-PEG-PLLA microparti-

cles (sample 12 in Table 2). The major peak at

1,758.20 cm-1 is corresponding to stretching vibration of

the C=O of PLLA-PEG-PLLA in Fig. 5a. While the major

peaks at 2,767.43 and 2,078.64 cm-1 in Fig. 5d are the

characteristic peaks of morphine, which may result from

the N–H symmetric and asymmetric stretching vibration in

the tertiary amine ion. The existence of the peaks at

1,647.70 and 1,619.79 cm-1 is the stretching vibration of

C=C alkene of the hexatomic ring. The peak at

1,505.55 cm-1 is the stretching vibration of C=C of the

aromatic ring [34]. The characteristic peaks of components

of aromatic ring and hexatomic ring were found in the

spectra of morphine and PLLA-PEG-PLLA physical mix-

ture in Fig. 5c. After SEDS process, the peaks in Fig. 5b at

2,078.01, 1,644.70, 1,617.78 and 1,505.48 cm-1 consisting

with the peaks of morphine can still be observed, indicating

that morphine indeed existed in the microparticles.

Besides, the peaks between 1,505 and 756 cm-1 were

strengthened, as well as the peaks at 2,998.64 and

2,944.51 cm-1, all benefiting from the introduction of

morphine. So it was evident that the drug was successfully

encapsulated and the SEDS process had no effect on the

main bonds of components.

3.3 XRD analysis

XRD analyses of unprocessed and processed drug-loaded

microparticles were performed to evaluate the eventual

structural changes at the crystal level. Figure 6 showed the

XRD patterns of morphine, PLLA-PEG-PLLA raw mate-

rials, morphine and PLLA-PEG-PLLA physical mixture

and morphine-loaded PLLA-PEG-PLLA microparticles

(sample 12 in Table 2). As shown in Fig. 6a, there were

several feature crystalline peaks of morphine in the range

of 5�–30�. The strong peaks in Fig. 6d between 10� and 25�can be regarded as the feature crystalline peaks of PLLA

and PEG in the PLLA-PEG-PLLA triblock copolymer. For

the physical mixture of morphine and PLLA-PEG-PLLA,

almost all characteristic crystalline peaks of morphine and

Fig. 5 FTIR spectrum of raw

materials and microparticles:

(a) PLLA-PEG-PLLA raw

materials, (b) morphine-loaded

PLLA-PEG-PLLA

microparticles, (c) morphine

and PLLA-PEG-PLLA physical

mixture, (d) morphine

Fig. 6 XRD patterns of raw materials and microparticles: (a) mor-

phine, (b) morphine-loaded PLLA-PEG-PLLA microparticles,

(c) morphine and PLLA-PEG-PLLA physical mixture, (d) PLLA-

PEG-PLLA raw materials


123

PLLA-PEG-PLLA can still be seen in Fig. 6c, though their

slight weakness in intensity. When referring to morphine-

loaded PLLA-PEG-PLLA microparticles, all the crystalline

peaks of morphine disappeared in Fig. 6b. So it was

demonstrated that the morphine existed in the morphine-

loaded PLLA-PEG-PLLA microparticles were amorphous

after SEDS process. This difference can be readily

explained by the very fast precipitation during SEDS pro-

cess which did not allow the organization of the compound

in a crystalline. The crystalline peaks of PLLA-PEG-PLLA

also weakened, which was in agreement with the previous

results [35].

3.4 In vitro morphine release properties

of microparticles

The morphine release profiles of PLLA and PLLA-PEG-

PLLA microparticles precipitated with different PEG

content in copolymer were shown in Fig. 7. The profiles of

PLLA-PEG-PLLA microparticles with 3 and 5 % PEG

consist of burst release followed by a sustained release.

While the PLLA microparticles and PLLA-PEG-PLLA

microparticles with PEG content of 1 % presented sus-

tained release throughout the process. Microparticles with

the highest PEG content of 5 % showed a 28.92 % burst

release in the first 0.5 h which was far more rapid than

14.93 % release of microparticles with 3 % PEG. Here

T80 % refers to the time when the cumulative release per-

centage of morphine reached about 80 %. It can be seen

from Fig. 7 that the T80 % of microparticles with PEG

content of 3 and 5 % were about 48 and 24 h, respectively.

The T80 % of PLLA microparticles was more than 696 h,

while the T80 % of microparticles with 1 % PEG even

reached 1,200 h. It was concluded that morphine release

profiles could be rationalized by optimizing the content of

PEG in the polymer matrix. The release mechanism of

drugs from polymeric devices made of poly-a-hydroxy-

acids and their derivatives involve diffusion or dissolution

of the drug and polymer degradation [10]. For some

authors, three different phases were described to explain

the release behaviors of microparticles: initial diffusion,

matrix hydration and degradation [13, 36]. The initial dif-

fusion resulted from drugs trapped on the surface of the

polymer matrix or loosely mosaic in the polymeric matrix

during the manufacturing process [36, 37]. So burst release

was observed in the initial incubation time. Then water

penetrated into the polymeric matrix through diffusion and

the encapsulated drug was released into the aqueous

medium. Finally, the degradation of polymeric matrix

performed the thorough release of drug in the microparti-

cles. Figure 7 implied that the release of morphine from

microparticles with PEG content of 3 and 5 % mainly

underwent the initial diffusion and matrix hydration pha-

ses. The release of morphine completed in several days

suggested that the degradation of polymer matrix did not

account for a major role. While microparticles with PEG

content of 1 % and PLLA microparticles went through all

three release phases. The PEG fragment introduced into the

PLLA-PEG-PLLA could facilitate the drug release because

of its hydrophilic and non-hydrolysable. It might act as

‘water pump’ inside the matrix. So it was the fact that

morphine released from microparticles seemed to be

mostly governed by PEG content inside the copolymer

(Fig. 7). Besides, the release rate of the microparticles

increases with molecular weight of the matrix copolymer

decreasing. A possible reason for this may be that lower

molecular polymers could provide more hydrated active

sites for hydrolysis and produced less diffusion resistance

for drug compared with higher molecular ones [38]. With

the constant PEG (MW = 2,000), the higher the content of

Fig. 7 Drug release profiles of

morphine-loaded PLLA-PEG-

PLLA microparticles with

different PEG contents in

PLLA-PEG-PLLA


123

PEG in PLLA-PEG-PLLA is, the lower the molecular

weight of polymer is. So considering all factors above,

microparticles with PEG content of 5 % reflected the most

evident burst release compared with microparticles with

PEG content of 3 and 1 %. However, the burst release of

morphine was of great significance in the controlled release


samples immersed for 1, 2, 4

and 8 weeks in PBS: morphine-

loaded PLLA microparticles

(left), morphine-loaded PLLA-

PEG (3 %)-PLLA

microparticles (right). The

insets are higher magnification

images corresponding to the

ellipse area


123

systems. As a drug used for analgesia of post-operative and

moderate to severe cancer pain, immediate-release and

extended-release behaviors are recommended [19, 20].

That’s to say, an initial burst release for analgesia followed

by prolonged release to promote gradual healing is desir-

able here. Several days or weeks controlled release systems

of morphine are preferred by many surgeons due to the

requirement for short-term treatment [39, 40]. While the

long phases release involved of drug levels below the

therapeutic level might lead to possible development of

drug resistance. The microparticles with 1 % PEG have a

release even lower than the PLLA microparticles, which

might result from the higher molecular weight and larger

particle size of PLLA-PEG(1 %)-PLLA microparticles [38,

41]. Besides, the PEG proportion is so small that the

improvement of PEG in the drug release is limited.

3.5 In vitro degradation properties

Polymer degradation is achieved by random hydrolysis of

the backbone chain. The rate of hydrolysis depends on

several factors such as polymer composition, molecular

weight, hydrophilicity, crystallinity and amorphous state.

Besides, the morphology and structure can also affect the

degradation when the polymer is formulated as micropar-

ticles for drug delivery system [10, 42, 43]. The degrada-

tion process of the PLLA microparticles began with a

decrease of molecular weight as a consequence of the

hydrolysis of ester bonds of the polymer when incubated in

PBS buffer. Water penetrates through the microparticles

originating smaller polymer chains, thus resulting in mass

loss and gradually morphology or structure changes [44].

Figure 8 showed the surface morphology of microparticles

after incubated into degradation media for 1, 2, 4 and

8 week. The original microparticles showed an overall

intact outer surface, while tiny pores were noticed after the

first week. The inset clearly showed the pores on the sur-

face of the microparticles and pore size was\200 nm. The

appearance of pores might result from the early dissolution

of hydrophilic morphine in the microparticles accompanied

by a heterogeneous degradation occurred on the surface of

polymer matrix even though it was not obvious [45]. More

pores formed and the size of pores increased apparently

after 2 weeks. The size of some pores almost reached about

400 nm shown in the inset of Fig. 8. Most of the micro-

particles appeared a certain degree of erosion and porous

through slow degradation. Morphological changes were

obvious which could be observed 4 weeks later. The deg-

radation occurred in the external and internal of micro-

particles at the same time and complex porous structures

were formed. So the mechanical strength of the micro-

particles were greatly weakened and it was hard to main-

tain their spherical morphologies, resulting in irregular

morphologies [11]. To sum up, the morphology changes of

the degraded microparticles can be divided into three

stages as shown in Fig. 9. At the first stage, several pores

were formed on the microparticles, then the number of

pores increased and the size of pores became larger. At the

third stage, the morphologies gradually became irregular.

Besides, the molecular weight (Mn) of PLLA-PEG-PLLA

microparticles analysed by 1H-NMR decreased from

6.77 9 104 to 5.93 9 104, which also indicated the bio-

material degradation.

By comparing the degradation morphologies at certain

time, it can be found that the degradation rate of PLLA-

PEG-PLLA microparticles was faster than that of PLLA

microparticles, which may result from the introduction of

hydrophilic PEG. Some authors demonstrated that there

were three phases in the PLLA-PEG-PLLA polymer sys-

tem when immersed in aqueous phase: hydrophobic PLLA

phase with the lowest water content, a swollen PEG phase

with the highest water content and a phase consisting of

PLLA and PEG with a higher water content than the PLLA

phase [29, 46]. So water penetrated through the micro-

particle matrix and preferentially stayed in PEG domains,

which was in favor of the hydrolysis of PLLA-PEG-PLLA

tri-block copolymer. Together with the excellent biocom-

patibility of PEG, the degradable and biocompatible

Fig. 9 Schematic diagram of

microparticle degradation

process


123

characteristics of PLLA-PEG-PLLA make it a potential

carrier for controlled delivery system.

4 Conclusion

Morphine-loaded PLLA and PLLA-PEG-PLLA micropar-

ticles were successfully prepared by the SEDS process. The

influence of process parameters including the ratio of

morphine to PLLA-PEG-PLLA, content of PEG in PLLA-

PEG-PLLA, pressure and concentration of morphine on

morphology, particle size, DL, encapsulation efficiency

and in vitro release properties were investigated. All the

microparticles presented spherical or ellipsoidal morphol-

ogy with the mean diameter of 2.04–5.73 lm. The DL of

microparticles was strongly dependent on the dosing ratio

of morphine to PLLA-PEG-PLLA. The highest DL of

17.92 % was obtained when the dosing ratio increased to

1:5. The encapsulation efficiency of microparticles can be

as much as 87.31 % at a morphine to PLLA-PEG-PLLA

ratio of 1:10, a PEG content of 3 %, a pressure of 12 MPa

and a morphine concentration of 8 mg/mL. The release

behaviors of microparticles varied greatly with the PEG

content in the PLLA-PEG-PLLA copolymer, showing

short-term release with burst release followed by sustained

release within days or long-term release lasted for weeks.

The degradation test indicated that the introduction of PEG

contributed to the faster degradation rate of PLLA-PEG-

PLLA compared with PLLA and a promising candidate for

delivery system could be envisioned.

Acknowledgments This work has been supported by the National

Natural Science Foundation of China (project No. 51173120,

51273122 and 51202151). Authors are very much grateful to the

National Engineering Research Center for Biomaterials, Sichuan

University for the assistance with the microscopy work.

References

1. Agnihotri SA, Aminabhavi TM. Novel interpenetrating network

chitosan-poly(ethylene oxide-g-acrylamide) hydrogel micro-

spheres for the controlled release of capecitabine. Int J Pharm.

2006;324:103–15.

2. Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, tox-

icity, biocompatibility and clinical applications of polylactic acid/

polyglycolic acid copolymers. Biomaterials. 1996;17:93–102.

3. Kawashima Y, York P. Drug delivery applications of supercriti-

cal fluid technology. Adv Drug Del Rev. 2008;60:297–8.

4. Kang Y, Yang C, Ouyang P, Yin G, Huang Z, Yao Y, Liao X.

The preparation of BSA-PLLA microparticles in a batch super-

critical anti-solvent process. Carbohydr Polym. 2009;77:244–9.

5. Chen AZ, Li Y, Chau FT, Lau TY, Hu JY, Zhao Z, Mok DKw.

Microencapsulation of puerarin nanoparticles by poly(L-lactide)

in a supercritical CO2 process. Acta Biomater. 2009;5:2913–9.

6. Shekunova YB, Baldygab J, York P. Particle formation by mix-

ing with supercritical antisolvent at high Reynolds numbers.

Chem Eng Sci. 2001;56:2421–33.

7. Bristow S, Shekunov T, Shekunov BY, York P. Analysis of the

supersaturation and precipitation process with supercritical CO2.

J Supercrit Fluids. 2001;21:257–71.

8. Vert M, Li S, Garreau H. More about the degradation of LA/GA-

derived matrices in aqueous media. J Control Release. 1991;

16:15–26.

9. Mothe CG, Drumond WS, Wang SH. Phase behavior of biode-

gradable amphiphilic poly(L, L-lactide)-b-poly(ethylene glycol)-

b-poly(L, L-lactide). Thermochim Acta. 2006;445:61–6.

10. Dorati R, Genta I, Colonna C, Modena T, Pavanetto F, Perugini

P, Conti B. Investigation of the degradation behaviour of

poly(ethylene glycol-co-D, L-lactide) copolymer. Polym Degrad

Stab. 2007;92:1660–8.

11. Zhou S, Deng X. In vitro degradation characteristics of poly-DL-

lactide–poly(ethylene glycol) microspheres containing human

serum albumin. React Funct Polym. 2002;51:93–100.

12. Park SJ, Kim SH. Preparation and characterization of biode-

gradable poly(L-lactide)/poly(ethylene glycol) microcapsules

containing erythromycin by emulsion solvent evaporation tech-

nique. J Colloid Interface Sci. 2004;271:336–41.

13. Duvvuri S, Janoria KG, Mitra AK. Development of a novel for-

mulation containing poly(D, L-lactide-co-glycolide) microspheres

dispersed in PLGA-PEG-PLGA gel for sustained delivery of

ganciclovir. J Control Release. 2005;108:282–93.

14. Hiemstra C, Zhong ZY, Van Tomme SR, Hennink WE, Dijkstra

PJ, Feijen J. Protein release from injectable stereocomplexed

hydrogels based on PEG-PDLA and PEG-PLLA star block

copolymers. J Control Release. 2006;116:e19–21.

15. Venkatraman SS, Jie P, Min F, Freddy BYC, Leong-Huat G.

Micelle-like nanoparticles of PLA-PEG-PLA triblock copolymer

as chemotherapeutic carrier. Int J Pharm. 2005;298:219–32.

16. Andersen G, Christrup L, Sjøgren P. Relationships among mor-

phine metabolism, pain and side effects during long-term treat-

ment: an update. J Pain Symptom Manage. 2003;25:74–91.

17. Polard E, Le Corre P, Chevanne F, Le Verge R. In vitro and

in vivo evaluation of polylactide and polylactide-co-glycolide

microspheres of morphine for site-specific delivery. Int J Pharm.

1996;134:37–46.

18. Moolenaar F, Meyler P, Frijlink E, Jauw TH, Visser J, Proost H.

Rectal absorption of morphine from controlled release supposi-

tories. Int J Pharm. 1995;114:117–20.

19. Eliot L, Butler J, Devane J, Loewen G. Pharmacokinetic evalu-

ation of a sprinkle-dose regimen of a once-daily, extended-release

morphine formulation. Clin Ther. 2002;24:260–8.

20. Portenoy RK, Sciberras A, Eliot L, Loewen G, Butler J, Devane J.

Steady-state pharmacokinetic comparison of a new, extended-

release, once-daily morphine formulation, AvinzaTM, and a

twice-daily controlled-release morphine formulation in patients

with chronic moderate-to-severe pain. J Pain Symptom Manage.

2002;23:292–300.

21. Alvarez-Fuentes J, Fernandez-Arevalo M, Holgado MA, Carab-

allo I, Rabasco AM, Mico JA, Rojas O, Ortega-Alvaro A. Pre-

clinical study of a controlled release oral morphine system in rats.

Int J Pharm. 1996;139:237–41.

22. Morales ME, Gallardo Lara V, Calpena AC, Domenech J, Ruiz

MA. Comparative study of morphine diffusion from sustained

release polymeric suspensions. J Control Release. 2004;95:

75–81.

23. Kang Y, Wu J, Yin G, Huang Z, Liao X, Yao Y, Ouyang P, Wang

H, Yang Q. Characterization and biological evaluation of pac-

litaxel-loaded poly(L-lactic acid) microparticles prepared by

supercritical CO2. Langmuir. 2008;24:7432–41.

24. Holgado MA, Iruin A, Alvarez-Fuentes J, Fernandez-Arevalo M.

Development and in vitro evaluation of a controlled release for-

mulation to produce wide dose interval morphine tablets. Eur J

Pharm Biopharm. 2008;70:544–9.


123

25. Tozuka Y, Miyazaki Y, Takeuchi H. A combinational super-

critical CO2 system for nanoparticle preparation of indomethacin.

Int J Pharm. 2010;386:243–8.

26. Marra F, De Marco I, Reverchon E. Numerical analysis of the

characteristic times controlling supercritical antisolvent micron-

ization. Chem Eng Sci. 2012;71:39–45.

27. Reverchon EM, DE Marco L. Mechanisms controlling super-

critical antisolvent precipitate morphology. Chem Eng J.

2011;169:358–70.

28. Reverchon E, Della Porta G, Sannino D, Ciambelli P. Super-

critical antisolvent precipitation of nanoparticles of a zinc oxide

precursor. Powder Technol. 1999;102:127–34.

29. Yang YY, Wan JP, Chung TS, Pallathadka PK, Ng S, Heller J.

POE-PEG-POE triblock copolymeric microspheres containing

protein: I Preparation and characterization. J Control Release.

2001;75:115–28.

30. Franceschi E, De Cesaro AM, Feiten M, Ferreira SRS, Dariva C,

Kunita MH, Rubira AF, Muniz EC, Corazza ML, Oliveira JV.

Precipitation of b-carotene and PHBV and co-precipitation from

SEDS technique using supercritical CO2. J Supercrit Fluids.

2008;47:259–69.

31. Chen AZ, Pu XM, Kang YQ, Liao L, Yao YD, Yin GF. Study of

poly(L-lactide) microparticles based on supercritical CO2. J Mater

Sci Mater Med. 2007;18:2339–45.

32. Boutin O, Badens E, Carretier E, Charbit G. Co-precipitation of a

herbicide and biodegradable materials by the supercritical anti-

solvent technique. J Supercrit Fluids. 2004;31:89–99.

33. Moshashaee S, Bisrat M, Forbes RT, Nyqvist H, York P.

Supercritical fluid processing of proteins: I: lysozyme precipita-

tion from organic solution. Eur J Pharm Sci. 2000;11:239–45.

34. Alnajjar AO, El-Zaria ME. Synthesis and characterization of

novel azo-morphine derivatives for possible use in abused drugs

analysis. Eur J Med Chem. 2008;43:357–63.

35. Sui X, Wei W, Yang L, Zu Y, Zhao C, Zhang L, Yang F, Zhang

Z. Preparation, characterization and in vivo assessment of the

bioavailability of glycyrrhizic acid microparticles by supercritical

anti-solvent process. Int J Pharm. 2012;423:471–9.

36. Batycky RP, Hanes J, Langer R, Edwards DA. A theoretical

model of erosion and macromolecular drug release from biode-

grading microspheres. J Pharm Sci. 1997;86:1464–77.

37. Huang X, Brazel CS. On the importance and mechanisms of burst

release in matrix-controlled drug delivery systems. J Control

Release. 2001;73:121–36.

38. Mallarde D, Boutignon F, Moine F, Barre E, David S, Touchet H,

Ferruti P, Deghenghi R. PLGA-PEG microspheres of teverelix:

influence of polymer type on microsphere characteristics and on

teverelix in vitro release. Int J Pharm. 2003;261:69–80.

39. Griffith LG. Polymeric biomaterials. Acta Mater. 2000;48:

263–77.

40. Zhao C, Kim SW, Yang DY, Kim JJ, Park NC, Lee SW, Paick JS,

Ahn TY, Min KS, Park K, Park JK. Efficacy and safety of once-

daily dosing of udenafil in the treatment of erectile dysfunction:

results of a multicenter, randomized, double-blind, Placebo-

Controlled Trial. Eur Urol. 2011;60:380–7.

41. Panyam J, Dali MM, Sahoo SK, Ma W, Chakravarthi SS, Amidon

GL, Levy RJ, Labhasetwar V. Polymer degradation and in vitro

release of a model protein from poly(D, L-lactide-co-glycolide)

nano- and microparticles. J Control Release. 2003;92:173–87.

42. Li S, Garreau H, Vert M. Structure-property relationships in the

case of the degradation of massive poly(a-hydroxy acids) in

aqueous media. J Mater Sci Mater Med. 1990;1:198–206.

43. Park TG. Degradation of poly(lactic-co-glycolic acid) microspheres:

effect of copolymer composition. Biomaterials. 1995;16:1123–30.

44. Blanco MD, Sastre RL, Teijon C, Olmo R, Teijon JM. Degra-

dation behaviour of microspheres prepared by spray-drying

poly(D, L-lactide) and poly(D, L-lactide-co-glycolide) polymers.

Int J Pharm. 2006;326:139–47.

45. Gopferich A, Langer R. Modeling of polymer erosion. Macro-

molecules. 1993;26:4105–12.

46. Youxin L, Volland C, Kissel T. In-vitro degradation and bovine

serum albumin release of the ABA triblock copolymers consist-

ing of poly (L(?) lactic acid), or poly(L(?) lactic acid-co-glycolic

acid) A-blocks attached to central polyoxyethylene B-blocks.

J Control Release. 1994;32:121–8.


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