Random L-lactide/e-caprolactone copolymers as drug deliverymaterials

Annalisa Dalmoro • Anna Angela Barba •

Marina Lamberti • Mina Mazzeo • Vincenzo Venditto •

Gaetano Lamberti

Received: 12 March 2014 / Accepted: 9 May 2014 / Published online: 24 May 2014

� Springer Science+Business Media New York 2014

Abstract In this work, the degradation phenomena and

the release kinetics of an active molecule from matrices

systems made of random copolymers of e-caprolactone

(CL) and L-lactide (LA) were investigated by exposing the

matrices, shaped as thin films, to simulated physiological

environments. a-tocopherol was incorporated into the films

as hydrophobic model molecule with the aim to investigate

both its release pattern and its effect on erosion phenom-

ena. In particular, the films have been kept at controlled

conditions (temperature, stirring, pH) and they were char-

acterized in terms of weight loss, water uptake, thermal

properties, and change of number average molecular

weight, in order to explain the molecule release kinetics

and the degradation pathways of the copolymers. The main

findings of this study are that the erosion phenomena take

place significantly only when a critical value of the

molecular mass was obtained in the sample; that the pre-

sence of the drug stabilizes the matrix and it decreases the

rate of molecular mass decrease; and that crystallinity,

reducing the chain mobility, causes lower erosion rates.

Introduction

The main class of synthetic biodegradable polymers is

represented by aliphatic polyesters [1]. These latter, syn-

thesized by ring opening polymerization (ROP) of lactones

(CL) and lactides (LA), is largely used for bioresorbable

devices in surgery (orthopedic devices, sutures, stents,

tissue engineering, and adhesion barriers) and for con-

trolled drug delivery, mainly for their good mechanical

properties, hydrolyzability, and biocompatibility [2, 3].

The method of ring opening polymerization was refined to

obtain polyesters with controlled architecture and tailor-

made properties with the aim to satisfy requirements of

pharmaceutical industries [3]. Polylactide (PLA) and

polycaprolactone are interesting for their properties: PLA

has in vivo shorter half-life (few weeks) than PCL (1 year);

in addition the PCL permeability to drugs is higher than

PLA. Alternative structures to the homopolyesters (linear

random, block copolymers) were investigated to combine

these properties. Co-polymerization of lactide and e cap-

rolactone was, therefore, tested with the aim to take

advantage of the degradability of PLA and permeability to

drugs of PCL [4]. Investigations were focused on the

effects of composition, crystallinity, molecular weight,

shape (surface extension and thickness), and drug loading

both on structural properties and on biodegradability [5],

even if the literature showed contradictions about these

effects by comparisons made among different co-polymer

molecular weights and compositions [6].

Degradation of aliphatic polyesters involves many dif-

fusion/reaction phenomena: water uptake, ester hydrolysis,

diffusion, and solubilization of soluble species. In vivo

degradation of PCL, PLA, and their random copolymers was

qualitatively the same: degradation rate of random copoly-

mers was much higher than those of the homopolymers

A. Dalmoro � A. A. Barba (&)

Dipartimento di Farmacia, DIFARMA, Universita degli Studi di

Salerno, via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy

e-mail: aabarba@unisa.it

M. Lamberti � M. Mazzeo � V. Venditto

Dipartimento di Chimica e Biologia, DCB, Universita degli

Studi di Salerno, via Giovanni Paolo II, 132, 84084 Fisciano,

SA, Italy

G. Lamberti

Dipartimento di Ingegneria Industriale, DIIn, Universita degli

Studi di Salerno, via Giovanni Paolo II, 132, 84084 Fisciano,

SA, Italy

123

J Mater Sci (2014) 49:5986–5996

DOI 10.1007/s10853-014-8317-x

under the same conditions. On the other hand, the degra-

dation rate of block copolymers was found intermediate

between those of PCL and PLA [2].

Due to the relevant potentiality of copolymers based on

LA and e CL in biomedical and pharmaceutical applica-

tions, many studies are currently on going on their degra-

dation phenomena in physiological environment. In

particular, among the many formulations for drug delivery,

polymeric films, especially with thin sheets (thickness of

200 nm–1 lm), are preferred for their slower degradation

and larger surface area [7] in contact with buffer solutions

used to mimic physiological environments. Films of

poly(DL-lactide-co-glycolide) added with an antiprolifera-

tive drug, paclitaxel, were tested for developing coated

coronary stents or fully biodegradable stent matrices [8].

Films of size 14 9 14 9 0.08 mm3 were obtained. Paclit-

axel release from these films showed a three-stage release

pattern: a slow diffusional release followed by an accel-

erated diffusion-degradation release stage and finally a

saturation stage. Wang et al. [9] studied the in vitro

5-Fluorouracil release from films (20 9 10 9 0.2 mm3) of

a co-polymer of polycaprolactone/poly-(ethylene oxide)/

polylactide (PCEL). Copolymers with a similar content of

poly-(ethylene oxide), PEO, but with a different LA/CL

ratios, showed mechanical and morphological properties

depending on LA/CL ratio. The degradation of PCEL

copolymers, thus, depended on composition and cristal-

linity. An amorphous and LA-rich co-polymer degraded

more easily than a crystalline or CL-rich co-polymer.

5-Fluorouracil release was function of its hydrophilic nat-

ure and the type of polymeric system. Huang et al. [4]

synthesized block copolymers PLA–PCL–PLA with a

molecular weight of about 60,000 a.m.u. and a crystallinity

lower than the homopolymer PCL owing to the LA seg-

ments. Films of size 10 9 10 9 0.4 mm3 were subjected

to degradation tests up to 60 weeks: they slowly absorbed

water for 18 weeks, then water uptake and mass erosion

increased up to 25 weeks, unlike PCL homopolymer,

which showed negligible erosion phenomena due to its

hydrophobic and more crystalline nature. Moreover, the

co-polymer LA/CL ratio, constant in the first 18 weeks,

decreased up to the disappearance of LA over 40 weeks,

with a consequent enhancement of crystallinity. Films of

random poly(lactide-co-glycolide (PLGA) (53 % rac-lac-

tide and 47 % glycolide) and PLLA copolymers were also

investigated to evaluate the effect of the drug chemical

nature on matrix degradation, using lidocaine in both base

and salt forms [6]. Films of both copolymers, with size

40 9 25 mm2 and a thickness of 25 lm, subjected to

degradation tests, showed that lidobase accelerated matrix

degradation. Also in this case, co-polymer crystallinity

highly influenced drug release profiles: release rates from a

crystalline matrix are slower if compared to the amorphous

counterpart having a similar molecular weight. In [10]

synthesis and characterization studies of low-molecular

weight random poly(L-lactide-co-e-caprolactone (PLCL)

copolymers and following degradation studies of micro-

sphere scaffolds of the same copolymers were carried out.

It was found that lower crystallinity and more ester groups,

with increasing LA content, had caused higher water

absorption and thus larger degradation rate. Moreover,

degradation tests of nanoparticles, based on amphiphilic

block copolymers of poly(caprolactone)–poly(ethylene

glycol)–poly(lactide) (PCELA), highlighted that the poly-

meric chemical composition was the key factor not only to

control nanoparticle size, yield, and drug-entrapment effi-

ciency, but also drug release behavior [11]. Therefore,

delivery systems based on polyesters seem to show release

properties strictly dependent on composition, thickness,

molecular weight, and crystallinity.

In this work studies on degradation behavior of films

made by random copolymers of L-LA and e-CL, at dif-

ferent LA/CL ratios, synthesized by novel zinc complexes

[12], were performed.

a-Tocopherol was incorporated into the films as

hydrophobic model molecule with the aims to investigate

its release pattern and the effect on erosion phenomena. In

particular, loaded and not loaded film-samples were kept at

controlled conditions (temperature, stirring, pH) that

mimicked the action of physiological environments and,

then, were characterized in terms of weight loss, water

uptake, and change of average molecular weight. As sug-

gested by literature, these parameters play a key role in

molecule release kinetics and in degradation pathways of

the copolymers.

Materials and methods

Materials

Hexane and toluene (Carlo Erba Reagents Sas, Cornaredo,

Milan, IT) were purified by distillation from sodium ben-

zophenone ketyl. e-CL (Sigma Aldrich Srl, Milan, IT) was

dried with CaH2 for 24 h at room temperature and then

distilled under reduced pressure. L-Lactide (Sigma Aldrich

Srl, Milan, IT) was purified by crystallization from dry

toluene. iPrOH (Sigma Aldrich Srl, Milan, IT) was purified

by distillation over sodium. (iPr)–PPP–Zn–N(SiHMe2)2

was prepared according to published procedures [12].

a-Tocopherol, TC, (Sigma Aldrich Srl, Milan, IT; Mw

430.71 a.m.u.; Stokes radius 0.49 nm), was used as model

molecule. Its uses are widespread, e.g., TC is added to

polyolefin as stabilizer during manufacturing, antioxidant

in polyesters for food packaging based [13]. Moreover, TC

shows relevant potentiality as active molecule due to its

J Mater Sci (2014) 49:5986–5996 5987

123

role in enzymatic activities, gene expression, and neuro-

logical functions. It was administered as antiinflammatory

agent, protector against cellular lipid peroxidation in car-

tilage to sustain the normal bone growth and modeling, and

as promoter of calcium retention and mechanical properties

in bone tissue [14].

Common solvents such as chloroform and dichloro-

methane, and all other chemicals and, i.e., monobasic

sodium phosphate and sodium hydroxide (Sigma Aldrich

Srl, Milan, IT), were commercially available and used as

received unless otherwise stated.

Methods

Copolymer synthesis

All manipulations of air and/or water-sensitive compounds

were carried out under dry nitrogen atmosphere using a

Braun Labmaster glovebox or standard Schlenk line tech-

niques. Glassware and vials used in the polymerizations

were dried in an oven at 120 �C overnight and exposed to

vacuum-nitrogen cycle, three times.

Random copolymers of L-LA and CL were synthesized

by ROP using (iPr)–PPP–Zn–N(SiHMe2)2 as catalyst. The

co-polymerization was carried out in inert atmosphere. In a

typical experiment, in a Braun Labmaster glovebox, a flask

equipped with a magnetic stirring bar was charged with a

toluene solution (0.5 mL) of the metal-complex (12 lmol),iPrOH (24 lmol), L-Lactide and e-caprolactone at defined

ratios ([e-CL ? L-LA]/[Zn] = 1400). The reaction mixture

was stirred for 120 min at 110 �C. After this time, a small

aliquot of the reaction mixture was sampled and dissolved

in about 0.5 mL of CDCl3. The resulting solution was

analyzed by 1H NMR spectroscopy to evaluate the con-

version of the two monomers. The reaction mixture was

cooled at room temperature and the product was dissolved

in CH2Cl2 and precipitated in n-hexane, then filtered and

dried under vacuum oven at 40 �C for 16 h.

Copolymers characterization

1H and 13C NMR analysis The NMR spectra were recor-

ded on Bruker Avance 400 spectrometer (1H, 400.00 MHz;13C, 100.62 MHz) at 298 K. Deuterated solvents were

purchased from Cambridge Isotope Laboratories, Inc. and

degassed and dried over activated 3 A molecular sieves

prior to use. Chemical shifts (d) are listed as parts per

million and coupling constants (J) in hertz. 1H NMR

spectra are referenced using the residual solvent peak at d7.27 for CDCl3. 13C NMR spectra are referenced using the

residual solvent peak at d = 77.23 for CDCl3.1H NMR of PCL-co-PLLA (400 MHz, CDCl3, 25� C):

1.31–1.47 (m, 2H, CH2 backbone), 1.48–1.51 (m, 3H,

C(O)CHCH3), 1.52–1.70 (m, 4H, CH2 backbone), 2.27 (t,

2H, C(O)CH2), 2.42 (m, 2H, C(O)CH2), 4.01 (t, 2H,

C(O)OACH2), 4.22 (t, 2H, C(O)OACH2), and 5.0–5.18 (m,

1H, C(O)CHCH3). 13C NMR of PCL-co-PLLA (100 MHz,

CDCl3, 25 �C): 16.88–17.18 (C(O)CHCH3), 24.53–24.69,

25.43–25.62, 28.39, 28.52, 33.91–34.26, 64.36, 65.29, 68.43

(O–CO–(CH2)5–), 68.43–69.46 (C(O)CHCH3), 125.50, 128.

43, 129.25, 169.85–170.0(C(O)CHCH3), 170.33–170.53

(C(O)O, CL–LA–LA), 170.26–170.31 (C(O)O, CL–LA–

CL), 170.38 (C(O)O, LA–LA–CL), 173.06(C(O)O, LA–

CL–LA), 173.08 (C(O)O, CL–CL–LA), 173. 70 (C(O)O,

LA–CLCL), and 173.73 (C(O)O, CL–CL–CL).

DSC Glass transition temperatures (Tg), melting points

(Tm), and melting enthalpies (DHm) of polymers were

measured by differential scanning calorimetry (DSC) using

a DSC 2920 apparatus manufactured by TA Instruments

under a nitrogen flux of 50 mL/min with a heating and

cooling rate of 10 �C min-1 in the range -100 to 200 �C.

All calorimetric data were reported for the second heating

cycle, unless otherwise stated.

Crystallinity degrees (XC) were calculated from melting

enthalpies using the equilibrium enthalpy of fusion (the

enthalpy for a 100 % crystalline sample) of PLA and PCL

homopolymers, 93.1 J/g [15] and 142.3 J/g [16], respectively.

GPC The molecular weights (Mn and Mw) and the

molecular mass distribution (Mw/Mn) of polymer samples

were measured by gel permeation chromatography (GPC) at

30 �C, using THF as solvent, flow rate of eluent 1 mL/min,

and narrow polystyrene standards as reference. The mea-

surements were performed on a Waters 1525 binary system

equipped with a Waters 2414 Refractive Index detector

using four Styragel columns (range 1000–1000,000 A).

Every value was the average of two independent measure-

ments, and it was corrected by a factor 0.58 [17].

Preparation of films

Thin films of LA/CL copolymers were obtained by the

technique of casting from polymer solution. About 0.5 g of

each co-polymer (LA/CL = 10/90; LA/CL = 50/50; LA/

CL = 70/30; and LA/CL = 90/10) was dissolved in about

20 g of chloroform. This solution was poured in a Teflon

plate (diameter: 6.1 cm), and the solvent was allowed to

evaporate in order to obtain a dry polymeric film having

thickness in the range 115–146 lm (by the thickness gauge

Kafer: 0.001–2 mm). Using the same procedure, TC-loa-

ded films were prepared using the ratio TC/copoly-

mer = 0.25. In particular, a-tocopherol (available in gel

form) was added to the solution polymer/solvent before the

solvent evaporation stage. An accurate stirring of the final

5988 J Mater Sci (2014) 49:5986–5996

123

mixture was performed to ensure a homogenous distribu-

tion of the a-tocopherol.

Degradation study

Six samples of size 15 9 15 mm2 were cut from each all

the prepared films (made with both unloaded and loaded

copolymers) and subjected to characterization and in vitro

degradation. Each sample was weighed and photographed

before its immersion in phosphate buffer solution (10 mL)

at pH 7.4 and its insertion in transparent vials with the aim

to also control the appearance modifications during the

degradation test. The vials were subjected to agitation

using an orbital shaker at 60 rpm (SSL1 Stuart) placed in a

thermostatic refrigeration chamber (DAS 37005 Intercon-

tinental) to have a temperature of 37 �C. Selected pH and

temperature simulate physiological conditions. The time-

line consisted of six sample taking: 7 days (1 week);

21 days (3 weeks); 35 days (5 weeks); 56 days (8 weeks);

84 days (12 weeks); and 196 days (28 weeks).

After each sample taking, the samples were subjected to

the following characterization, such as evaluation of water

uptake, weight loss, molecular weight decrease, and

thickness variation, as well as detection of a-tocopherol

release (for loaded films).

Water uptake and weight loss

Samples were withdrawn from the bulk, wiped, photo-

graphed, and weighted. Then, samples were dried to con-

stant weight in order to determine the weight loss. Water

uptake and weight loss were calculated at each time by the

following equations:

Water� uptake ð%Þ ¼ Ww �Wd

Wd

� 100 ð1Þ

Weight� loss ð%Þ ¼ Wi �Wd

Wi

� 100; ð2Þ

where Ww, Wd, and Wi represent the wet weight, dry

weight, and initial weight of the samples, respectively.

Drug release assay

The a-tocopherol release was detected for the loaded

samples subjected to the degradation study. In a typical

test, after the sample withdrawal at the predetermined time,

a volume of 1 mL of buffer solution was removed, diluted

with 9 mL of ethanol, and centrifuged at 3000 rpm for

5 min (centrifuge R-8C Remi) with the aim to extract the

lipophilic tocopherol from the buffer solution. The amount

of drug released was quantitatively estimated using an UV–

VIS spectrometer (Lambda 25 Perkin Elmer). The full

absorption spectra were collected in a wavelength range

from 200 to 400 nm, since the height of a-tocopherol peak

is close to 285 nm, to avoid incorrect measurements due to

shift in kmax. The procedure of spectra subtraction, instead

of the simple reading of the absorbance at a given wave-

length, was used to eliminate the interferences due to

polymers or other substances. It consisted in a subtraction

of an exponential curve from the spectrum in the range of

absorbance of the drug (in this case between 275 and

325 nm), more details are reported in [18]. Briefly, the

curve obtained was fitted with a Gaussian curve, optimiz-

ing its parameters using the least squares methods: in this

way, the parameter that replaced the absorbance value, to

be considered for the measure of drug concentration, was

the height of the fitted Gaussian curve. Obviously, the

proportionality constant necessary to correlate absorbance

to concentration, was obtained by a calibration curve where

the ordinate values were not the absorbance values, but the

heights of the fitted Gaussian curves at given concentra-

tions. Finally, to close the mass balance on a-tocopherol, a

part of the dried sample was weighed and dissolved in

dichloromethane: the solution was centrifuged and sub-

jected to spectrometer analysis to detect the residual

amount of a-tocopherol in samples after the relevant deg-

radation time.

Results and discussion

PLCL copolymers: synthesis and characterization

Generally, in the copolymerization reactions LA is a more

reactive monomer than e-CL, therefore, the most of metal

catalysts are able to produce only polymer chains with a

gradient or blocky distribution from LA to CL. Random

copolymers have been previously obtained with traditional

catalytic systems such as Sn(Oct)2 by randomization pro-

cesses promoted by side transesterification reactions [10].1

This method, even if simple and efficient, does not allow a

strict control of the copolymer composition and the

molecular weights, parameters that are fundamental for the

design of drug delivery systems.

Recently, we introduced a new class of pincer-type zinc

complexes able to promote the random polymerization of

L-LA and e-CL. The structures obtained copolymers were

not a result of secondary processes of transesterification,

but a consequence of ability of the catalyst to incorporate

efficiently both the monomers [19–22].

1 Transesterification is the process of exchanging the alkoxy groups

between polymer chains. There are two types of transesterification

reaction: intramolecular leading to formation of cyclic oligomers and

intermolecular leading to an increase in the range of chain lengths.

J Mater Sci (2014) 49:5986–5996 5989

123

In this work, PLCL copolymers synthesized with dif-

ferent compositions LA/CL = 10/90; LA/CL = 50/50;

LA/CL = 70/30; LA/CL = 90/10) are used. Copolymeri-

zation tests were carried out by totally 1400 equivalents of

the two monomers to obtain polymers with the same

molecular weighs. For all reactions, the percent conversion

of lactide was quantitative, while the percent conversion of

e-caprolactone ranged from 85 to 99 % (Table 1). The

chemical compositions of the obtained copolymers were

determined by 1H and 13C NMR spectroscopy. For all

samples, the percentages of lactide and e-caprolactone

units in the polymer chains, calculated by 1H spectra, were

coherent with the monomer feed ratio, thus demonstrating

the good propensity of the selected catalytic system to

produce copolymers of the designed composition. By 13C

NMR analysis of the polymer microstructure was excluded

the occurrence of transesterification reactions.

Moreover, for all samples, molecular weights distribu-

tions are mono-modal, with narrow polydispersity indexes

(PDI in a range between 1.48 and 2.06).

Heat properties (Tg, glass transition temperature; Tm,

melting temperature; DHm, enthalpy of melting) of as-

polymerized samples are shown in Table 2. Heat properties

of PLA and PCL homopolymers, achieved by the same

catalytic systems, are also reported for comparison.

Copolymer glass transition temperatures increase line-

arly with LA % content from PCL to PLA according to

several reported studies on LA–CL random copolymers

[19, 23, 24], even if similar behavior is exhibited also by

block copolymers.

As generally occur for random copolymers, the melting

temperature decreases by increasing the comonomer con-

tent, so that the melting temperatures of the copolymers

LA/CL = 90/10 and 10/90 are, respectively, lower than

those of the two homopolymers. This behavior is due to the

fact that reducing the sequence lengths of single comono-

mers, size and imperfections of crystals increase as long as

the crystallization is prevented. As a matter of fact, the

sample with the LA/CL = 54/46 does not show any

melting temperature.

As regards the sample LA/CL = 70/30, the observed

melting temperature, lower than that of the LA/CL = 90/

10 sample, suggests that short sequences of LA units are

also able to crystallize, although the resulting crystallinity

is low (DHm = 1 J/g). This result is in good agreement

with data reported by Vanhoorne et al. [23] for random

LA–CL copolymers showing a certain crystallinity degree

even for samples with CL unit content up to 40 %.

The degradation tests, described in the following para-

graph, were performed on samples in form of films which

were obtained by casting from polymer solution. The

properties of these films depend on the polymer crystalli-

zation conditions during the solvent evaporation, for this

reason the thermal properties of the prepared casting films

are reported in Table 2. In particular, melting temperatures

and melting enthalpies of chloroform casting film samples,

evaluated from the first heating DSC run, are reported.

While melting temperatures of film samples are very

similar to those of as-polymerized samples, melting

enthalpies are, in some cases, very different. In particular,

the LA/CL = 70/30 copolymer shows a melting enthalpy

of 32.8 J/g when prepared in form of solvent casting film,

while as-polymerized sample has a melting enthalpy of 1 J/g.

This difference comes from the fact that the two samples

crystallize in different conditions, the as-polymerized

sample is crystallized from melt (the melting enthalpy is

evaluated on second heating DSC run), while the casting

film sample is crystallized from solvent evaporation.

In Table 2 the crystallinity degree (Xc) of chloroform

casting film samples are also reported.

Degradation study

Polymer dissolution in a solvent is essentially caused by

two phenomena: solvent diffusion within the polymer and

breaking of polymer chains [25]. Water solvent penetrates

in polymer matrix and hydrolyzes ester bonds, causing a

decrease in molecular weight [1]. In particular, even if

polyesters show a molecular weight reduction as soon as

they are placed in contact with water, their polymer chains

are still too long to diffuse outwards, whereas, for higher

Table 1 Results of LA–CL random co-polymerization

Sample # Conversion % LA % CL % MnGPC (9103) PDI

10/90 100/99 9 91 66.5 1.48

50/50 100/99 54 46 58.0 2.06

70/30 99/85 69 31 43.9 1.46

90/10 100/99 90 10 48.6 1.55

Table 2 Thermal properties of as-polymerized and casting film

samples

LA % Tga

(�C)

Tma

(�C)

DHma

(J/g)

Tmb

(�C)

DHmb

(J/g)

Xcb

(%)

0 -62.0 57.6 71.9 60.3 77.7 55

9 -47.9 39.1 43.3 44.5 56.8 40

54 4.0 No No No No 0

69 28.3 140.2 1.0 137.4 32.8 35

90 45.8 158.1 25.7 157.4 37.3 40

100 55.0 170.6 53.9 172.5 61.4 66

No not observed; thermal data of PLA and PCL homopolymers are

reported for comparisona As-polymerized samples, data from second heating DSC runb Chloroform solvent casting film samples, data from first heating

DSC run

5990 J Mater Sci (2014) 49:5986–5996

123

degradation times, shorter chains (oligomers) diffuse more

rapidly and cause a loss in weight sample [26]. A consistent

loss of polymer weight occurs only when a specific value

of molecular weight, named critical molecular weight, is

reached. This value depends on polymer kind: 1050–1150

a.m.u. for PLA [27], below 5000 a.m.u. for PCL [7], in the

range between 5000 and 15000 a.m.u. for PLGA [28]. So

the degradation of a polyester, and therefore of the syn-

thesized copolymers, can be schematized in three steps:

hydration, hydrolysis, and erosion.

In this study, degradation phenomena were performed

on the film-samples prepared (samples of 15 9 15 mm2

and 131 ± 15 lm in thickness) and aged (at pH 7.4 and

37 �C under gently stirring) as previously reported. For

each sampling (as established by the planned time line),

loaded and unloaded copolymers were subjected to the

evaluation of visual aspect, weight loss, water uptake, and

decrease of molecular weight to assess and understand the

erosive phenomena and to detect the molecule release.

Visual observation

Samples of LA/CL = 10/90, LA/CL = 70/30, and LA/

CL = 90/10 initially were opaque and stiff owing to their

crystallinity, whilst LA/CL = 50/50 displayed a transpar-

ent and rubber-like appearance. All the samples became

opaque, whitish or yellowish for unloaded and loaded

copolymers, respectively, during degradation, and finally

became very fragile. Loaded copolymers kept their shape

up to 196 days, thanks to the stabilizing effect of a-

tocopherol, while unloaded samples began to break up after

56 days for the LA/CL = 70/30 co-polymer, and after

84 days for the remaining copolymers. An example of

evolution in visual aspect is shown for LA/CL = 70/30 and

LA/CL = 50/50 unloaded and loaded samples in Fig. 1

(photos are referred to the six unloaded and six loaded

samples taken after different times from the dissolution

bulk).

Water absorption, weight loss and molecular weight

changes

Degradation tests showed that both loaded and unloaded

LA/CL = 50/50 samples underwent to the greatest water

uptake and weight loss, as well as to the fastest molecular

weight decrease owing to its amorphous nature, different

from the semi-crystalline character of the other copolymers.

Moreover, the presence of a-tocopherol acted as stabilizer

Fig. 1 Evolution in visual

aspect during degradation test of

LA/CL = 70/30 loaded and

unloaded samples (up) and LA/

CL = 50/50 loaded and

unloaded samples (down)

J Mater Sci (2014) 49:5986–5996 5991

123

in loaded samples also thanks to its hydrophobic nature. The

water uptake for both loaded and unloaded copolymers for

the different days of immersion is shown in Fig. 2. As

expected, LA/CL = 50/50 and LA/CL = 50/50 ? TC

absorbed more water than other samples. LA/CL = 50/

50 ? TC seemed to absorb more water than LA/CL = 50/

50 in the last two sampling, in contrast with the hydro-

phobic contribution of TC, but it was due to the early dis-

integration of unloaded samples that caused a greater

difficulty in water uptake measurements. In fact, water

uptake data on loaded copolymers are more reliable thanks

to the more durable integrity of samples in immersion bulk.

The co-polymer with LA/CL ratio of 10/90, regardless of

TC presence, absorbed an insignificant amount of water

throughout the experimentation, according to its higher

content of CL (more hydrophobic) and semi-crystalline

feature. LA/CL = 70/30 and LA/CL = 90/10 showed a

similar trend, owing to their similar Mw and crystallinity. In

particular, they underwent a constant water uptake up to

196 days, when a significant increase occurred. However,

LA/CL = 90/10 showed a smaller water uptake at 196 days

than LA/CL = 70/30, and lower initial sample thickness

(134 versus 141 lm). This can be explained on the basis of

both the higher initial molecular weight (48.6 9 10-3 vs

43.9 9 10-3 a.m.u.) and its Tg (45.8 �C), which was the

only one above the test temperature (37 �C). Therefore,

polymeric chains were less sliding limiting the water uptake

and consequently hydrolysis. The difference in water

uptake was more pronounced for loaded samples (LA/

CL = 90/10 ? TC vs LA/CL = 70/30 ? TC) because

inclusion of tocopherol was shown to cause an increase in

crystallinity [29] and therefore a consequent reduction of

chains mobility, especially for LA/CL = 90/10 ? TC.

The water uptake behavior is closely related to molec-

ular weight variation, Mn, shown in Fig. 3. Even if the first

data show an unexpected increase in molecular weight,

probably due to experimental inconveniences, the signifi-

cant trends are of molecular weight reduction because of

the degradation. Some values after 196 days are not

available owing to the low-molecular weight, difficult to

observe by the instrument.

For unloaded samples (left, Fig. 3), copolymers with

LA/CL ratio of 50/50 were subjected to a quicker molec-

ular weight decrease, with respect to the other samples.

LA/CL = 50/50 molecular weight, after 84 days of

immersion, was about 5000 a.m.u., i.e., the critical value

for oligomers formation, that diffused more easily, causing

also a great weight loss [7]. Molecular weights of the other

three copolymers, LA/CL = 10/90, LA/CL = 70/30, and

LA/CL = 90/10, were still over the critical value after

84 days (21.2 9 103, 8.5 9 103, 11.2 9 103, respectively).

Molecular weights of LA/CL = 70/30 and LA/CL = 90/

10 showed the same trend, which were intermediate

between LA/CL = 50/50 (fast) and LA/CL = 10/90

(slow). Finally, after 196 days of dissolution test, all the

unloaded samples reached the critical molecular weight, as

visible by copolymers degradation and as proved by the

low-molecular weight, 4900 a.m.u., of the co-polymer LA/

CL = 10/90, which was the most resistant to degradation.

The presence of a-tocopherol (right, Fig. 3) prevented the

achievement of critical molecular weight for all the loaded

copolymers during the dissolution test. After 84 days, the

molecular weights of LA/CL = 50/50 ? TC, LA/CL = 10/

90 ? TC, LA/CL = 70/30 ? TC, and LA/CL = 90/

10 ? TC were 7.5 9 103, 28.7 9 103, 15.2 9 103, and

34.1 9 103, respectively. These Mn were all over the

Fig. 2 Change in water absorption profiles for the four copolymers, without a and with b drug loading, during degradation test

5992 J Mater Sci (2014) 49:5986–5996

123

threshold value and higher than values of unloaded samples.

After 196 days of immersion, LA/CL = 10/90 ? TC still

had a high-molecular weight (about 10000 a.m.u.), in

agreement with its behavior during degradation test: it was

subjected to the lowest water uptake and to the lightest ero-

sion. The behavior of LA/CL = 90/10 ? TC was more

similar to LA/CL = 10/90 ? TC: the greater Tg of LA/

CL = 90/10 ? TC affected hydrolysis time, and therefore,

oligomers loss, making its response to erosive phenomena

similar to the more crystalline and hydrophobic LA/

CL = 10/90 ? TC. Molecular weight decrease and water

uptake trends were confirmed by erosion data as shown in

Fig. 4. Again, the LA/CL = 50/50 co-polymer underwent

the largest weight loss, that was more evident for unloaded

samples: after 84 days, unloaded co-polymer (left, Fig. 4)

showed a weight loss of 50 %, according to the achievement

of threshold Mn, while loaded sample (right, Fig. 4 lost the

25 % in weight because its molecular weight was still high.

After 84 days, the other both loaded and unloaded copoly-

mers had a molecular weight too high so that the erosion was

limited. At the end of the test (196 days), as previously

described, all the unloaded copolymers reached the critical

molecular weight. In particular, unloaded samples of LA/

CL = 70/30 and of LA/CL = 90/10 showed a substantial

weight loss, of 45 and 32 %, respectively, which was less

pronounced for LA/CL = 10/90 (about 10 %) for its more

crystalline nature and the higher content of CL. Loaded

copolymers, at the end of dissolution test, were all broken,

but they did not reached the critical molecular weight. LA/

CL = 70/30 ? TC was subjected to a weight loss of about

35 %, while LA/CL = 10/90 ? TC and LA/CL = 90/

10 ? TC showed the same erosion of about 5 %. Results

about the low weight loss of LA/CL = 90/10 ? TC after

196 days are in agreement with the smaller water uptake and

slower decrease of molecular mass.

The degradation of the copolymers in dissolution bulk

can be, therefore, described by three consecutive steps:

hydration; hydrolysis, which breaks the chains until the

attainment of a critical molecular mass with formation of

oligomers; and finally, erosion, caused by oligomers

leaving polymeric films. Thus, during hydrolysis the

molecules become small enough to begin losing weight,

since the small molecules dissolve in the aqueous med-

ium. In addition, the crystallinity can change during

degradation, and thus influence the degradation rate of the

polymer [30].

The kinetic equation describing the scission of polyester

chains during hydrolysis can be obtained by assuming that

scission is catalyzed by the terminal carboxyl group, with

the hydrolysis rate proportional to the concentration of

water and esters [31]. The hydrolysis constant, k, was

calculated with the following procedure:

d COOH½ �=dt ¼ k0 COOH½ � � H2O½ � esters½ �; ð3Þ

where [COOH] is the concentration of the terminal car-

boxyl group. If the product [H2O] [esters] is supposed to

be constant, by integrating the Eq. (3), coupled with the

relationship [COOH] � Mn - 1, the results is:

Mn;t ¼ Mn;0 exp �ktð Þ; ð4Þ

where Mn,t and Mn,0 are the number-average molecular

weight at hydrolysis time t and 0, respectively, and k is

given by the product k0[H2O] [esters]. Equation (4) can be

converted to the following equation:

ln Mn;t ¼ ln Mn;0 � kt ð5Þ

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100 LA/CL=50/50 LA/CL=10/90 LA/CL=70/30 LA/CL=90/10

Mol

ecul

ar w

eigh

t ×10

3

Time (days)

(a)

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120 140 160 180 2000

10

20

30

40

50

60

70

80

90

100

(b) LA/CL=50/50 + TC LA/CL=10/90 + TC LA/CL=70/30 + TC LA/CL=90/10 + TC

Mol

ecul

ar w

eigh

t ×10

3

Time (days)

0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200

0 20 40 60 80 100 120 140 160 180 200

Fig. 3 Molecular weight trend with time for unloaded a and loaded b samples

J Mater Sci (2014) 49:5986–5996 5993

123

Thus, k values are the slopes of the straight lines, having

‘‘ln(Mn,t/Mn,0)’’ as ordinate and ‘‘t’’ as abscissa, in Fig. 5

and they are shown in Table 3.

All the considerations done until now were confirmed by

the hydrolysis constant values:

• LA/CL = 50/50 showed the largest hydrolysis,

according to its amorphous nature, while hydrolysis

of the loaded polymer (LA/CL = 50/50 ? TC) was

delayed by the presence of a-tocopherol as

stabilizer;

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

(b) LA/CL=50/50 + TC LA/CL=10/90 + TC LA/CL=70/30 + TC LA/CL=90/10 + TC

Wei

ght l

oss

(%)

Time (days)

0

10

20

30

40

50

60

70

80

90

1000 20 40 60 80 100 120 140 160 180 200

0

10

20

30

40

50

60

70

80

90

100 LA/CL=50/50 LA/CL=10/90 LA/CL=70/30 LA/CL=90/10

Wei

ght l

oss

(%)

Time (days)

(a)

0 20 40 60 80 100 120 140 160 180 200

0 20 40 60 80 100 120 140 160 180 2000 20 40 60 80 100 120 140 160 180 200

Fig. 4 Weight loss percentage versus time for the four samples, without a and with b a-tocopherol during degradation test

-3

-2

-1

0

-3

-2

-1

0

(b)

LA/CL=50/50 + TC

LA/CL=10/90 + TC

LA/CL=70/30 + TC

LA/CL=90/10 + TC

ln(m

n/m

n0)

Time (days)

-3

-2

-1

0

0 20 40 60 80

0 20 40 60 80

0 20 40 60 80

0 20 40 60 80

-3

-2

-1

0

LA/CL=50/50

LA/CL=10/90

LA/CL=70/30

LA/CL=90/10

ln(m

n/m

n0)

Time (days)

(a)

Fig. 5 Semi-logarithmic plot of Mn against dissolution time for unloaded (left) and loaded (right) copolymers

Table 3 Hydrolysis constants, k, for samples with and without a-tocopherol

Unloaded co-polimer LA/CL = 50/50 LA/CL = 10/90 LA/CL = 70/30 LA/CL = 90/10

k 9 10-3 (days-1) 40.2 12.8 12.5 15.2

Loaded co-polimer LA/CL = 50/50 ? TC LA/CL = 10/90 ? TC LA/CL = 70/30 ? TC LA/CL = 90/10 ? TC

k 9 10-3 (days-1) 22.1 8.2 10 2.3

5994 J Mater Sci (2014) 49:5986–5996

123

• LA/CL = 10/90, LA/CL = 70/30, LA/CL = 90/10 had a

similar hydrolysis constant, confirming the erosion behav-

ior; the corresponding loaded polymers showed lower

k values owing to the stabilizing effect of a-tocopherol;

• LA/CL = 10/90 ? TC and LA/CL = 70/30 ? TC

exhibited a comparable k, while the hydrolysis constant

of LA/CL = 90/10 ? TC was unexpectedly the lowest

(because LA/CL = 10/90 ? TC should be less sub-

jected to hydrolysis for its high amount of hydrophobic

CL and crystalline nature). This delay of hydrolysis

phenomena for LA/CL = 90/10 ? TC was justified by

the higher co-polymer Tg, as previously discussed.

Release phenomena

The percentage of released a-tocopherol, defined as the

ratio between the TC mass measured in bulk at the sam-

pling time and the TC initial mass in the sample, was

evaluated during dissolution test, as shown in Fig. 6.

Copolymers released negligible amounts of TC during the

test, lower than 2–3 % as visible by zooming in Fig. 6. The

trend of released TC is in agreement with observations

about erosive phenomena because it occurred as a result of

copolymers erosion, which was anyway moderate. The

presence of the hydrophobic molecule stabilized the

copolymers structure, according to the delayed decrease of

molecular weight, slowing down hydrolysis and polymeric

chain scission, which became consistent only at the end of

dissolution step, after 196 days. In particular, the LA/

CL = 50/50 ? TC copolymer, even undergoing a high

loss weight of 55 %, released a small amount of a-

tocopherol, because it did not still reached the critical Mn.

Conclusions

In this work, random copolymers of LA/CL with different

ratios between LA and CL were synthesized and characterized

in terms of thermal properties (glass transition temperature,

melting temperatures, and enthalpy of fusion), as well as in

terms of number average molecular weight and molecular

weight distribution. In order to investigate their potential uses

to produce drug release systems, thin films of the different

synthesized copolymers were immersed in a fluid mimicking

the physiological environment (pH 7.4), and kept at physio-

logical temperature (37 �C) under gentle stirring, for long

times. The film samples were obtained by casting from

polymer solution, using the copolymers pure and loaded with

a hydrophobic molecule, the a-tocopherol, as a model drug.

The evolutions of film hydration (water up-take), polymer

erosion (weight loss), drug release, and molecular weight

were monitored for a period of 28 weeks. The hydration of

the films was faster for the amorphous polymers, and it has

caused the molecular weight decrease (mediated by the

hydrolysis of ester bonds), and, in turn, the erosion of the

films. The analysis of molecular weights allowed identifying

a critical value, roughly 5000 a.m.u., below which the

degradation of the films take place fast and at a full extent.

In the loaded films, the presence of the a-tocopherol

stabilizes the structures, delaying the hydration and the

erosion. The kinetic of the drug release is coherent with the

erosion phenomenon, thus it is possible to conclude that the

drug release is due only to erosion.

In conclusion, a proper selection of the LA/CL ratio, of

the crystallinity degree of the polymer, of the nature of the

drug and of the initial polymer molecular weight, was found

able to determine the erosion kinetics, and therefore, the

drug release kinetics. Hence, the proposed systems, based

on the random copolymers of LA/CL, in principle can be

used to design and realize systems with a tailored erosion,

and thus with drug release profiles.

Acknowledgements This work was supported by the Ministero

dell’Istruzione dell’Universita e della Ricerca-MIUR (contract grant

number PRIN 2010/2011-20109PLMH2). Annalisa Dalmoro’s

research grant was supported by ‘‘Strategie Terapeutiche Innova-

tive’’—STRAIN, POR Campania FSE 2007/2013. The Authors thank

dr Ilaria D’Auria for the GPC measurements.

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0

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