Evaluation of drug delivery characteristics of microspheres of PMMA–PCL–cholesterol obtained by...

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Journal of Controlled Relea

Evaluation of drug delivery characteristics of microspheres of

PMMA–PCL–cholesterol obtained by supercritical-CO2

impregnation and by dissolution–evaporation techniques

Carlos Elviraa,*, Alejandra Fanovichb, Mar Fernandeza, Julio Fraileb,

Julio San Romana, Concepcion Domingob

aDepartamento de Quımica Macromolecular, Instituto de Ciencia y Tecnologıa de Polımeros, CSIC, c/Juan de la Cierva 3,

28006 Madrid, SpainbInstituto Ciencia de Materiales de Barcelona, CSIC, Campus de la UAB, 08193 Bellaterra, Spain

Received 14 April 2004; accepted 30 June 2004

Available online 10 August 2004

Abstract

Poly(methyl methacrylate), PMMA, and of PMMA/Poly(q-caprolactone), PCL, microspheres were loaded with different

amounts of cholesterol by using a supercritical carbon dioxide (SC-CO2) impregnation process in order to use a clean technique

with the absence of organic solvents, and to provide information for the infusion of additives into nonporous polymeric

substrates. A conventional dissolution–evaporation method was also used to obtain PMMA and PMMA–PCL microparticles

loaded with cholesterol. The obtained microspheres were characterized by environmental scanning electronic microscope,

ESEM, nuclear magnetic resonance spectroscopy, NMR, and differential scanning calorimetry, DSC, thermal analysis. A

comparison of drug release from particles obtained using both methods, the supercritical and the conventional, is presented.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Supercritical fluids; Controlled release; Cholesterol; PMMA; Poly(q-caprolactone)

1. Introduction

In recent years, considerable attention has been

focused on the development of drug delivery systems,

which attempt to increase bio-availability, sustain,

localize or target drug action in the body. Polymer

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2004.06.020

* Corresponding author. Tel.: +34-9156-22900; fax: +34-9156-

44853.

E-mail address: [email protected] (C. Elvira).

microparticles are often employed as supports to

deliver drugs, either as microspheres (monolithic

devices) or microcapsules (reservoir devices). Current

techniques for preparing such microparticles use large

amounts of organic solvents. Because of cost and

environmental regulations associated to the use of

organic solvents, the pharmaceutical industry has been

moving away from the use of these solvents, and

alternative technologies have been developed, such as

the supercritical fluid (SCF) technology [1]. Small

se 99 (2004) 231–240

C. Elvira et al. / Journal of Controlled Release 99 (2004) 231–240232

changes in temperature and pressure near the critical

region induce dramatic changes in the density of

SCFs, thereby facilitating the use of environmentally

benign agents such as carbon dioxide (CO2) for their

solvent and antisolvent properties. Polymeric materi-

als have a very limited solubility in supercritical

carbon dioxide (SC-CO2), and this is an interesting

feature for producing monolithic loaded microspheres,

obtained by an impregnation process [2]. The

impregnation process is based on polymer swelling

caused by CO2, which facilitates both impurities

diffusion to the fluid phase and active agent infusion

into the polymeric phase. The polymers used in this

work are microspheres of poly(methyl methacrylate),

PMMA, and microspheres of PMMA with poly(q-caprolactone), PCL, prepared by suspension polymer-

ization of MMA in the presence of PCL. Cholesterol

has been considered as a solute-model compound

representative of low aqueous-solubility steroids.

PMMA is an acrylic hydrophobic biostable poly-

mer that is widely used in the biomedical field as bone

cement in orthopaedics and traumatology [3,4], and as

implant carrier for sustained local delivery of anti-

inflammatory or antibiotics drugs [5,6]. PCL is a soft

and hard tissue-compatible material that is used as a

bioresorbable matrix, for example, to prepare oily core

microcapsules containing bovine serum albumin for

the controlled delivery of protein vaccines [7], and as

a matrix material for tissue engineering [8]. Polymer

blends formulations can improve or at least maintain

the mechanical properties, diminish the toxicity,

provide a partial biodegradable character and control

the release of bioactive molecules. In this sense, new

polymer blends and composite materials have been

synthesized as PCL-b-PMMA crosslinked block

copolymer [9]; P(MMA-co-HEMA) hydrogel matri-

ces [10]; PLGA/PMMA nanoparticles [11]; biode-

gradable and stable composite foams of porous apatite

cement infiltrated with PCL or PMMA [12]; calcium

phosphates and PMMA composites [13]; glass/

PMMA composites [14]; and PMMA–PCL heteroge-

neous blends [15]. In our case, the combination of

biostable PMMA and biodegradable PCL polymers

provide materials with a good mechanical integrity,

which can be an alternative to traditional extrudable

ethylene vinyl acetate (EVA) copolymer, used in the

sustained release of bioactive compounds applied in

tissue regeneration processes [16].

On the other hand, an increasing number of newly

developed drugs are poorly soluble in water. Fur-

thermore, most bio-molecules have a very short half-

life in vivo. These compounds should be administered

immersed in a polymeric matrix. The effectiveness of

powdered drug systems is extremely sensitive to

particle size and specific surface area, with the

dissolution rate being dependent on these factors.

Hence, poorly soluble drugs should be obtained as

particulate systems of very small particle size.

However, for many materials the precipitation of

nanometric systems with the required characteristics is

not easy, or even possible, using conventional

technology. In general, by increasing the specific

surface area of a particulate system, the overall mass-

transfer coefficient and, hence, the dissolution rate of

the powder increases correspondingly. When nano-

metric particles cannot be obtained, the total surface

area available for dissolution can be enhanced by

adsorbing the active principle in a system, usually a

porous matrix. In a similar respect, nonporous swel-

lable matrixes can also be used. In general, when

crystallization is avoided, the molecules of solute are

more easily detached from a polymeric matrix than

from a crystal of the same composition of the solute.

The objective of this study is to advance in the

SCF-impregnation process, and to provide informa-

tion for the infusion of additives into nonporous

polymeric substrates. PMMA and PMMA–PCL poly-

mers in the form of microspheres are used as matrices

charged with different amounts of cholesterol. A

conventional method for preparing cholesterol-con-

taining PMMA and PMMA–PCL particles is also

described. Finally, a comparison of drug release from

particles obtained using both methods, the super-

critical and the conventional, is presented.

2. Materials and methods

2.1. Materials

Cholesterol (Ch, Fluka N99 wt.%) was chosen as

the organic solute to be impregnated. The selected

polymeric matrixes, poly(methylmethacrylate)

(PMMA: wt.%, Mw=12.2�105 g mol�1), poly(q-caprolactone) (PCL: Mw=66,500 g mol�1) and the

blends PMMA–PCL (85PMMA15PCL wt.%,) were

C. Elvira et al. / Journal of Controlled Release 99 (2004) 231–240 233

synthesized in our laboratory, following a published

procedure [15]. These prepared polymers contain

amounts of residual monomer MMA (about 4%-wt)

and initiator benzoyl peroxide, BPO (about 1%-wt).

The synthesis of PCL was carried out by ring-opening

polymerization of q-caprolactone. PMMA-based pol-

ymers in the form of microparticles (spherical beads)

or solution of PCL in MMA monomer were synthe-

sized by the suspension polymerization of MMA.

Water was used as suspension medium, poly(vinyl

alcohol) as suspension agent and BPO as a radical

initiator. Details of polymerization procedure, micro-

particle size and size distribution and morphology are

given in Ref. [15].

2.2. Methods

2.2.1. Supercritical microparticles impregnation

process

The behaviour of solute and polymers upon contact

with SC-CO2 was studied in a high-pressure one-pass

flow bench-scale equipment (Fig. 1). The equipment

included a saturator, S, an impregnation vessel, I. S

and I tubular vessels (ca. 10 ml) were held in an air-

oven (F1 K). CO2 was liquefied through a cooling

unit, CU, and compressed to the operating pressure

with a pump (Lewa EK-M-210). The fluid was pre-

heated to the desired temperature (Tad) in a heat

exchanger, HE, before entering the saturator. System

pressure (Pad) was controlled (F0.2 MPa) by means

of a back pressure regulator (Tescom 26-1761). The

Fig. 1. Equipme

saturator was loaded with a few grams of organic

solute and the impregnation vessel was loaded with ~1

g of polymer beads enclosed in cylinders made of

0.45 Am pore paper filter. During runs, the system was

maintained at a low mass flow rate (~0.5 SLM). A

typical impregnation experiment started by stabilizing

the system with CO2 during a period of 20–30 min.

Valve V1 and V2 were kept open and V3 closed. Then,

the CO2 was allowed to flow through the vessels for a

selected period of time. Valve V1 and V3 were kept

open and V2 closed. At the end of the flow period, the

system was depressurized. The impregnation experi-

ments were repeated three times to check the

reproducibility of the process. The impregnation gave

values with a deviation lower than 10% with respect

to the total loaded cholesterol.

2.2.2. Conventional microparticles formation process

One gram of PMMA or PMMA and PCL (85/15

w/w) polymers were dissolved in 12 ml of dichloro-

methane together with 10 or 20 wt.% of cholesterol.

The homogeneous solution was dispersed in 100 ml of

water with 1 wt.% of poly-(vinyl alcohol) and stirred

with an Ultra-Turrax at 8000 rpm for 30 min and then 3

h with magnetic stirring. Finally, the suspension was

centrifuged and the precipitated microparticles were

washed with distilled water.

2.2.3. Characterization

Purity and composition of processed solid samples

were determined by proton nuclear magnetic reso-

nt set-up.


nance (1H NMR) spectroscopy. 1H NMR spectra were

recorded in a Varian-300 spectrometer, in deuterated

chloroform solutions (5% (w/v)). Micrographs of

polymer beads were obtained by using an environ-

mental scanning electron microscope (ESEM, Phillips

XL30). The average diameter of the particles before

and after the impregnation process was determined by

image analysis of the corresponding ESEM micro-

graphs (Fig. 2a and b). The analysis program included

in the software of the ESEM equipment was used for

these experiments. The accuracy in the random

selection of microparticles and the average diameter

was adjusted to the measurement of more than 150

particles in different zones of the sample under

observation. Thermal transitions of the prepared

particles were analyzed by differential scanning

calorimetry (DSC) on Perkin-Elmer DSC-4. Typical

sample weights were 6–8 mg. Thermal transition

temperature measurements were conducted by heating

the samples from 303 to 473 K at 10 8C min�1,

rapidly cooling to 303 K and reheating to 473 K.

Glass transition temperature, Tg, and melting temper-

ature, Tm, were measured in both scans and taken as

Fig. 2. ESEM micrographs of (a) raw PMMA–PCL, (b) PMMA–PCL SC-C

(c) PMMA with 10% cholesterol, (d) PMMA–PCL with 10% cholesterol,

the peak in the case of Tm, and as the onset point of

the transition region of the Tg.

2.2.4. Cholesterol release measurements

An in vitro elution method was used to determine

the release behaviour of loaded formulations [17]. The

samples were immersed in vials containing 10 ml of

an ethanol/water (50:50 wt.%) solution and incubated

at 310 K without stirring. 0.5 ml of dissolution

medium was collected at different periods of time.

Fresh dissolution medium (0.5 ml) was then added for

the next period. The release measurements were

determined by means of high-performance liquid

chromatography (HPLC, Perkin Elmer LC-250) with

a Waters ABoundapack C-18 column. An UV–VIS

detector was employed (k=211 nm). An hexane/

isopropyl alcohol (70:30 wt.%) solution was used as

the mobile phase at a flow rate of 1 ml min�1. All

samples were assayed in triplicate. The retention time

of the cholesterol peak in samples, relative to the

standard, was 2.88F0.01 (n=200). The calibration

curve was made for the complete set of measurements,

displaying a correlation coefficient of 0.971.

O2 impregnated with cholesterol. Obtained by conventional method:

(e) PMMA–PCL with 20% cholesterol.


3. Results and discussion

The supercritical CO2 technology provides a clean,

cheap and friendly method for the extraction, purifi-

cation or impregnation of specific compounds into

selected supports. In particular, SC-CO2 is very

attractive for the treatment of polymeric supports

such as polyacrylic systems, biodegradable polyesters,

etc. The polymeric support is not soluble in super-

critical CO2, but in appropriate conditions of pressure

and temperature, CO2 behaves as a very low density

fluid that penetrates into the polymeric support by

diffusion giving rise to a relative swelling of the

system and loading the support with specific low

molecular weight compounds (drugs) soluble in the

SC-CO2 fluid. All the operation of diffusion and load

is produced in a relatively short time without

noticeable alteration of the structure and morphology

of the polymeric support.

3.1. SCF cholesterol impregnation of PMMA–PCL

particles by SC-CO2 method

For the SC-impregnation experiments, the influ-

ence of time, Pad and Tad in the uptake were

previously studied [18]. After process conditions

optimization, a Pad=20 MPa and a Tad=313 K were

chosen to perform the runs. Cholesterol was selected

as a model solute because its solubility in pure CO2 is

on the order of 0.1�10�4 mole fraction in the SC-

conditions of 20 MPa and 313 K [19]. For both

matrices, PMMA and PMMA–PCL, varying the

adsorption time from 2 to 10 h, different loads of

cholesterol were obtained. The average diameter of

the microspheres used in the SC-CO2 impregnation

process is exhibited in Table 1. These values

Table 1

Average diameter of microspheres used in the SC-CO2 impregna-

tion process, and obtained by the dissolution–evaporation conven-

tional method

Technique Sample Average diameter

(Am)

SC-CO2

impregnation

PMMA 14F8

PMMA–PCL 21F11

Dissolution–

evaporation

PMMA–CHOL10 3F1

PMMA–CHOL20 5F2

PMMA–PCL–CHOL10 10F5

PMMA–PCL–CHOL20 9F3

correspond to the average obtained by ESEM analysis

of particles before and after impregnation. It is clear

from Fig. 2a and b and the data reported in Table 1,

that the particles impregnated are somewhat bigger

than the original samples, but there was not observed

agglomeration, apparently. This means that the

impregnation process produces slight increase of the

size by the incorporation of the cholesterol in the bulk

of particles. It has to be considered that the final size

of particles and aggregates depends on the mode of

the depressurization process, and the conditions were

adjusted to be identical in all the experiments.

3.2. Particles loaded with cholesterol by conventional

method

The dissolution evaporation method used to obtain

PMMA and PMMA–PCL particles loaded with

different amounts of cholesterol was optimized using

the conditions described in the experimental part. As

the amount of loaded cholesterol was increased the

obtained microspheres showed remarkable deforma-

tions (pseudo spherical) and the spherical geometry

was lost, attributed to partial cholesterol crystalliza-

tion as needles inside of the polymeric matrixes are

formed. In this sense, maximum amount of loaded

cholesterol was fixed up to 20%-wt. This effect can be

observed in Fig. 2 where are shown the ESEM

micrographs of some microspheres obtained by this

method with different amounts of cholesterol. The

average diameter is also exhibited in Table 1.

3.3. Characterization

1H NMR spectra (Fig. 3) show that signals

corresponding to MMA (at 5.3 and 5.8 ppm) and

BPO (at 7.2 and 7.9 ppm) disappeared from the

polymer after SC-processing, i.e., the polymer was

purified as was previously observed [20]. Samples

processed with solute in the saturator displayed the

cholesterol signals with final loads of 10 (2 h

adsorption time) and 20%-wt (10 h adsorption time)

determined by the integration of the signals assigned

to –CH3 group (at 0.6 ppm) and –CH– (at 4.8 ppm) of

cholesterol. The ratio PMMA–PCL in the blend was

similar before and after SC-treatment as signals

integration of –OCH3 (at 3.5 ppm) from PMMA and

–CH2– from PCL (at 3.9 ppm) did not change.

Fig. 3. NMR spectra of PMMA–PCL: raw, purified and cholesterol

impregnated ( Pad=20 MPa, Tad=313 K, tad=20 h) using the SC-CO2

method.


Synthesized PMMA–PCL microspheres are a micro-

heterogeneous system, where semicrystalline PCL

domains are immersed in a matrix of amorphous

PMMA (Fig. 2a). For the PMMA–PCL SC-treated

Fig. 4. NMR spectra of raw cholesterol and PMMA–PCL particles charge

samples, beads deformation was not evident (Fig. 2b)

under working pressure and temperature. Fig. 4 shows

the 1H NMR spectra of raw cholesterol, PMMA–PCL

with 10%-wt and with 20%-wt of cholesterol prepared

following the conventional approach. The assignment

of the signals was performed as in the case of SC-CO2

impregnated particles, being the amounts of polymers

and cholesterol the same as described in the exper-

imental part. Microparticles prepared by the conven-

tional method (Fig. 2c–e) showed spherical and

pseudo-spherical geometry with dimensions between

2 and 15 Am of diameter as shown in Table 1. It can

be observed that particles of PMMA and cholesterol

have the smallest diameter, with a narrow particle size

distribution, whereas PMMA–PCL samples the aver-

age diameter increases up to 10F5 Am.

Table 2 shows the transitions obtained from the

DSC diagrams of the raw and loaded PMMA and

PMMA–PCL microparticles for the first and the

second scan. The three main transitions are assigned

to the PCL melt (Tm), the PMMA glass transition (Tg)

and the cholesterol melt (Tm). The melting temper-

ature of PCL does not change form the 1st to the 2nd

d with 20% and 10% of cholesterol using the conventional method.

Table 2

Thermal transitions of the prepared polymers (by SC-CO2 impregnation and by conventional methods) loaded with different amounts of

cholesterol

Method Sample % Cholesterol 1st scan (K) 2nd scan (K)

Tm

PCL

Tg

PMMA

Tm

Cholesterol

Tm

PCL

Tg

PMMA

Tm

Cholesterol

Raw 326 397 423

SC-CO2

impregnation

PMMA10 10 – 391 Not observed – 383 Not observed

PMMA20 20 – 392 Not observed – 384 Not observed

PMMAPCL10 10 328 391 Not observed Not observed 385 Not observed

PMMAPCL20 20 329 392 Not observed Not observed 383 Not observed

Conventional PMMA10 10 – 389 407 – 372 Not observed

PMMA20 20 – 388 415 – 373 412

PMMAPCL10 10 327 390 419 328 373 Not observed

PMMAPCL20 20 327 393 416 329 364 412


scan. In the case of PMMA, Tg decreases in the 2nd

scan and when increasing the amount of cholesterol in

the prepared particles. Cholesterol melting transition

was only observed for larger loadings in micro-

particles obtained using the conventional method.

For samples prepared by SC-impregnation, no choles-

Fig. 5. Cholesterol release profiles in ethanol/water 50:50, (a) SC

terol melting peak was observed. The appearance of

the cholesterol melting peak, and the PMMA Tg

remarkable reduction in the 2nd scan (ca. 20 K) of the

conventionally prepared samples was attributed to the

plasticization effect provoked by cholesterol mole-

cules (higher when increasing its percentage) which

-impregnated formulations, (b) conventional formulations.


initially are crystallized and, after melting in the 1st

scan, are diffused in the amorphous state acting as a

plasticizer of the PMMA matrix. In the case of SC-

impregnated samples, the absence of the melting

cholesterol peak, and lower temperature reduction of

the PMMA Tg in the 2nd scan, was attributed to the

formation of stable cholesterol–PMMA intermolecu-

lar interactions with the absence of crystallized

cholesterol. The Tg PMMA decrease is also influ-

enced by the presence of PCL, that as have been

observed in our laboratory, is an indicative of a very

poor compatibility between both polymers which are

segregated readily in two phases [15].

3.4. Drug release profiles

Drug release profiles in 50:50 ethanol/water media

are shown in Fig. 5 for all the formulations. The total

release of cholesterol of the SC-impregnated samples

(Fig. 5a) was reached in ca. 100 h in formulations

Fig. 6. Cholesterol release profiles in ethanol/water 50:50. Comparison

(b) formulations of PMMA–PCL.

with only PMMA. In the case of formulations with

PMMA and PCL, the total release of cholesterol was

reached in ca. 250 h. The presence of PCL in these

SC-impregnated formulations caused a significant

decrease in the release rate without differences

between samples with 10 and 20%-wt of cholesterol.

The release profiles shown in Fig. 5a also exhibit

great differences in cholesterol release being very fast

(90% in 100 h) in the case of PMMA formulations,

whereas in PMMA–PCL samples, an initial burst

effect is observed releasing the 35% of cholesterol in

100 h, and then a slow and sustained release in several

steps.

Conventionally prepared samples (Fig. 5b) showed

an initial sustained release of ca. 50%-wt in about 100

h for the formulation of PMMA with 10%-wt. This

release rate decreased significantly in formulations of

PMMA with 20%-wt of cholesterol. In the same

manner as for SC-formulations, the presence of PCL

decrease the rate release in all cases but this reduction

between the methods of preparation: (a) formulation of PMMA,


is more important in formulations with 20%-wt of

cholesterol. The lower release rate in formulations

with 20%-wt of cholesterol can be attributed to the

presence of crystallized cholesterol as was observed in

the DSC analysis.

When comparing the release profiles of similar

formulations (Fig. 6) obtained by different preparation

methods (conventional and SC-CO2) it can be

observed that these are similar in the case of PMMA

samples with exception of PMMA loaded with 20%

of cholesterol conventionally prepared (see Fig. 6a).

In the case of PMMA–PCL samples, the profiles are

also similar, faster when lower the amount of

cholesterol, and several release steps are observed in

all formulations (see Fig. 6b).

The different release steps observed was likely due

to different discharge behaviour of adsorbed/amor-

phous and crystallized cholesterol. In general, the

cholesterol was faster released as the presence of

crystalline cholesterol was lower. The faster release

observed in the SC-impregnated sample could be

attributed to liberation of cholesterol molecules

adsorbed in the polymeric matrix, but not crystallized.

The drug release was quantified by using the

Power law [21]:

Mt=Minf ¼ Ktn ð1Þ

where Minf is the absolute cumulative amount of drug

released at infinite time (which should be equal to

absolute amount of drug incorporated within the

system at time t=0), K is a constant characteristic of

the drug/polymeric system and n is the release

exponent, indicative of the mechanism of drug

release. The release behaviour of all formulations

was anomalous (non-Fickian) with n values in the

range of 0.43–0.85 for sphere polymeric controlled

systems.

The results of the in vitro release of cholesterol

from all formulations illustrated that the percent

release rate decreased proportionally to drug loading

and PCL fraction. In addition, this process occurred a

little faster for the drug-impregnated microparticles

(SC) than for the drug-crystallized microparticles

(conventional), following similar release profiles

when prepared by the two different techniques, fast

and one step in PMMA samples, and slower and

several steps in PMMA–PCL formulations.

4. Conclusions

SC-CO2 plasticizes PMMA-based polymeric

matrices. Plasticization was accompanied by swelling

with a consequent increase in the free volume of the

polymer that was responsible for the enhanced

diffusion of solute molecules in and out such systems.

It was shown that CO2 removed toxic residual

monomers and initiator of polymerization from

PMMA-based polymers, opening the possibility of

more biomedical applications. CO2 accelerated the

adsorption of low molecular weight additives, like

cholesterol. Applying a conventional method to obtain

similar microparticles, the cholesterol crystallized

inside of the matrix. Faster release rates were obtained

in systems where the cholesterol was impregnated

than in systems where the cholesterol was crystallized,

and the same type of release profiles were obtained in

the same polymer samples independently of the

preparation method applied (conventional or SC).

The impregnated monolithic systems can be used to

enhance the bio-availability of low-water solubility

drugs, and the supercritical CO2 technology provides

a useful way to purify the polymer matrix and load the

drug simultaneously.

Acknowledgment

The authors wish to acknowledge EC Project

Suprophar G1RD-CT-2000-00164 for financial sup-

port, to Programa Ramon y Cajal and to Comunidad

de Madrid.

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