International Journal of Pharmaceutics 342 (2007) 72–77

Influence of end groups on in vitro release and biological activity of

lysozyme from a phase-sensitive smart polymer-based in situ gel
forming controlled release drug delivery system
Sumit Chhabra, Vishal Sachdeva, Somnath Singh ∗
Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE 68178, USA
Received 12 January 2007; received in revised form 13 April 2007; accepted 30 April 2007
Available online 6 May 2007

Abstract
Phase-sensitive in situ gel forming controlled release formulations of lysozyme were prepared using poly lactic acid (PLA) and/or poly glycolic
acid (PGA) based polymers differing in end groups in addition to composition, and a solvent system consisting of various ratios of benzyl benzoate
(BB) and benzyl alcohol (BA). The amount of lysozyme in the released samples was determined by measuring absorbance at 280 nm using suitable
controls to nullify the effect of absorption of formulation degradation products. Biological activity of lysozyme was studied by an enzyme activity
assay using Micrococcus lysodeikticus as substrate. Polymers bearing carboxylic acid end group were not soluble in 100% BB but polymers having
ester end groups were soluble up to 27% (w/v) except polymer 4. A biphasic release profile consisting of slower first phase followed by faster
second phase was observed. Formulations prepared from polymer with carboxylic acid groups showed significantly (p < 0.05) lower burst release
(4%) than those containing ester end groups (20–30%). However, formulations consisting of polymer with carboxylic acid end groups showed
significantly (p < 0.05) faster release rate of incorporated lysozyme, although the total amount released was less in comparison to the total amount
released from formulations prepared using polymers containing ester end groups. The mean percentage specific enzyme activity (MPSEA) data
were supported by the release profiles. In conclusion, polymer end groups may influence the release profiles of a protein from an in situ gel depot
forming controlled release formulations.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Phase sensitive; Smart polymer; End groups; Lysozyme

1. Introduction As a consequence, increasing attention has been focused on

methods for administering these biologically active agents con-
Proteins possess unique physical and chemical properties tinuously at a sustained rate for an extended period of time.
which create difficulties in formulation and delivery. With the The primary method of accomplishing controlled release has
tremendous growth of biotechnology and sequencing of the been through incorporating the biologically active agents within
human genome, a large number of therapeutically active pro- polymers.
teins are being developed (Lee, 2002). Proteins are susceptible In situ gel forming, injectable phase-sensitive, smart poly-
to degradation by gastric enzymes and acidic environment, first mer systems have been found as promising controlled drug
pass metabolism, and pulmonary proteases activity; therefore, delivery systems (Al-Tahami et al., 2006; Singh and Singh,
they are mainly administered by parenteral route. Since most of 2004; Hatefi and Amsden, 2002). The injectable gel systems
protein drugs are cleared rapidly from systemic circulation, they which utilize a polymeric matrix and a solvent/solvents, have
must be given frequently and in high dose which may cause toxi- been investigated which takes advantage of solvents differing
city and meet poor patient compliance (Robinson and Talmadge, in their relative hydrophilicity/hydrophobicity (Okumu et al.,
2002). 2001). The hydrophilic solvents used by various researchers
are benzyl alcohol, N-methyl pyrrolidone, and ethanol, and
the hydrophobic solvents used are benzyl benzoate, miglyol,
∗ Corresponding author. Tel.: +1 402 280 3548; fax: +1 402 280 3320. and triacetin (Cleland et al., 2001). The hydrophilic solvent
E-mail address: ssingh@creighton.edu (S. Singh). leaves the gel quickly upon injection while the hydropho-

0378-5173/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2007.04.034
 S. Chhabra et al. / International Journal of Pharmaceutics 342 (2007) 72–77 73

bic solvent leaves the gel very slowly in comparison to a a model protein lysozyme from in situ gel forming controlled
hydrophilic solvent. Consequently, a shell that is spongy and release formulations.
porous forms rapidly around the exterior of the gel with a
hydrophilic solvent; while a shell also forms with the hydropho- 2. Material and methods
bic solvent, but it is smoother and has much smaller pores
which slows water penetration to the gel thereby decreasing 2.1. Materials
the rate of polymer degradation by hydrolysis and controlling
the release of the incorporated protein (Chandrashekhar et al., All the polymers ranging in IV from 0.19–0.32 dl/g
2001; Cleland et al., 2001; Duenas et al., 2001; Eliaz and Kost, [(Resomer® R 202 S, 202 H, and 203 S, and Resomer® RG 502 S
2000; Ravivarapu et al., 2000a,b). The polymers used must be and 502 H] were purchased from Boehringer Ingelheim (Peters-
biodegradable and biocompatible, e.g. polylactic acid, polygly- burg, VA, USA) and used as obtained without any purification.
cholic acid, poly lactic-co-glycholic acid, poly ␧-caprolactone, Lysozyme (L6876) and Micrococcus lysodeikticus ATCC# 4698
etc. (M3770) were purchased from Sigma–Aldrich, Inc. (St. Louis,
The functional groups residing at the end of these polymers MO, USA). BB and BA were purchased from Acros Organics,
may influence the release profiles of the incorporated proteins. New Jersey, USA. All other chemical used were of analytical
However, there is a little information available regarding the reagent quality.
influences of these end groups on the various parameters con-
trolling the release and stability of the incorporated/entrapped 2.2. Methods
proteins. Polymer end groups influence surface properties of
polymers which could be critical for rate of release of drugs from 2.2.1. Preparation of injectable polymer solution
nanoparticles and microspheres (Jalbert et al., 1997). Polymer Different concentrations (5–27%, w/v) of each polymer
end groups could affect the sensitivity of chemical amplifica- (Polymers 1–5) were added to different solvent mixtures in a
tion resist systems based on acid catalysis in a way altogether glass vial and kept in a shaking water bath (37 ◦ C, 35 rpm) for
different from the conventional resist systems (Ito et al., 1992). 24 h. Solubility was determined by visual inspection. Injectabil-
A patented surface modifying end groups (SMEs) technology ity of the polymer solution was determined by passing through
can generate desired surface chemistry without even using sur- a 22-gauge needle. The solvent mixtures were prepared by mix-
face modifying additives which sometimes have limitations in ing two solvents (BB and BA) in different ratios (100:0–60:40,
generating the desired surface properties (Ward and White, respectively). Those polymer solutions were chosen for the
1996). study, which were easily injectable at 37 ◦ C through a 22-gauge
End group modification in addition to, drug loading, polymer needle.
molecular weight, and copolymer composition, is reported to be
a critical factor affecting the in vitro/in vivo release of a drug from 2.2.2. Preparation of polymer–drug solution
nanoparticles. The intravenous administration of monomethoxy Injectable polymer solutions were mixed with a fixed con-
(polyethylene glycol)-poly (lactide-co-glycolide)-monome- centration (5%, w/v) of protein (lysozyme) and homogenized
thoxy (polyethyleneglycol) [mPEG-PLGA-mPEG] nanoparti- (5000 rpm, 1 min). Eight formulations differing in polymer
cles of mitoxantrone in mice resulted in prolonged mitoxantrone composition, end groups, intrinsic viscosities, and solvent
residence in systemic blood circulation compared to the intra- composition were prepared by following the steps shown in
venous administration of PLGA nanoparticles without the Fig. 1.
presence of methoxy end groups (Duan et al., 2006). End group
modification is also reported to influence the release of the 2.2.3. In vitro release
drug from a multilayered PLGA stent matrix. The release of Five hundred microliters of the formulation were injected
sirolimus from a bi-layer polymeric film used in stent was into 10 ml of isotonic phosphate buffer (pH 7.4) kept in a glass
found to decrease when the drug-eluting layer was made from vial. The formulation immediately changed into a gel depot. The
PLGA bearing a lauryl ester end group instead of acid group vials containing in situ formed gel depot were kept in a recipro-
(Wang et al., 2006). cal shaking water bath (Precision Scientific, Winchester, VA) at
In an in situ forming PLGA based microparticle formulation 37 ◦ C and 35 rpm for the entire period of study. One milliliter of
of leuprolide, PLGA with free carboxylic acid end groups led aliquot was withdrawn at specified time points which were used
to a lower drug release compared to PLGA with esterified end for determining quantity, biological activity, and conformational
groups (Luan and Bodmeier, 2006). End group is also found to stability of lysozyme. The volume withdrawn was replaced with
influence encapsulation efficiency of a drug in a microsphere fresh releasing media.
formulation. In a PLA and PLGA based microspheres higher
encapsulation of huperzine was found when the polymer was 2.2.4. Quantification by UV–vis spectrophotometer
containing free carboxylic acid group at the end of polymer One milliliter of the suitably diluted sample/standard was
chain (Gao et al., 2006). used for measuring the absorbance in UV–vis spectrophotome-
Therefore, in this study we have investigated the influence ter (UV 1700, Shimanzu, Kyoto, Japan) at 280 nm. Samples
of carboxylic acid and ester groups at the end of PLA/PLGA from formulation without lysozyme were used as blank control
based polymer on the rate of release and biological activity of for absorbance. Amount of lysozyme released in the samples
74 S. Chhabra et al. / International Journal of Pharmaceutics 342 (2007) 72–77

(Shugar, 1952):
Units of lysozyme (ml sample)
(A450 nm / min Test − A450 nm / min Blank) × df
=
(0.001)(0.1)
where df = dilution factor; 0.001 = change in absorbance at A as
per the unit definition; 0.1 = volume (in ml) of sample/standard
used.

2.2.6. Data analysis

Statistical comparisons were made using student’s t-test and
analysis of variance (ANOVA). The level of significance was
used as p < 0.05.

3. Results and discussion

3.1. Polymer solubility in various solvent systems

Table 1 lists the characteristics and solubility of the polymers

used in this study. Solubility of polymers was determined in a
solvent system consisted of different ratios of BB and BA. A
mixture of BB and BA provides an easy tool to manipulate to
obtain a solvent system of optimal hydrophilicity or hydropho-
bicity for a particular polymer. Polymers 2 and 3 were found
Fig. 1. Preparation of polymer–drug solution. insoluble in BB which might be due to their relative hydrophilic-
ity provided by the presence of acidic end groups because all
other polymers were found in BB.
was obtained from the standard curve and corrected for sample Polymers 1, 4 and 5 were relatively hydrophobic in nature
removal (Hayton and Chen, 1982). due to the presence of ester end groups and therefore were
soluble in BB. The maximum solubility of polymers 1 and 5
observed was 27% (w/v) but for polymer 4, it was 20% (w/v)
2.2.5. Biological activity of lysozyme by enzyme activity in BB which might be due to polymer composition. Polymer 4
assay is consisted of PLA and GA (50:50) but polymers 1 and 5 are
Suitable amount of Micrococcus lysodeikticus cell suspen- entirely consisted of PLA only (Table 1 and Fig. 2a and b). Con-
sion (0.015%, w/v) was prepared by using mixing Micrococcus sequently, polymer 4 could be expected to be less hydrophobic
lysodeikticus (Sigma Chemicals, St. Louis, MO) in potassium than polymer 1 or 5. Therefore, polymer 4 gets solubilized to
phosphate buffer (pH 6.24 at 25 ◦ C). Two and a half milliliters of a lesser extent (20%) in comparison to polymer 1 or 5 (27%)
this cell suspension were taken into a spectrophotometeric cell, in BB, a more hydrophobic solvent than BA (Merck Index,
and hundred microliters of an appropriated diluted lysozyme 2001),
sample (200–400 lysozyme units/ml) was added to it. The rate Polymer 2 demonstrated greater solubility in the solvent sys-
of decrease in absorbance at 450 nm was monitored by a UV tem containing mixture of BB and BA (70:30), while polymer
spectrophotometer (UV 1700, Shimanzu, Kyoto, Japan) dur- 3 favored the two solvents in a ratio of 30:70 which too can
ing the total period of 3 min. Number of biologically active be explained on the basis of polymer composition. Polymer 3
lysozyme units were determined by using following formula consists of PGA in addition to the PLA but polymer 2 is com-

Table 1
Characteristics of polymers used for preparing sustained release formulations

Polymer Composition Intrinsic viscosity (dl/g) Hydrophilicitya Degradationa End groups Solvent system (BB:BA) Concentrationb (w/v%)

Polymer 1 PLA* 0.22 Low Slow Ester 100:00; 70:30 27

Polymer 2 PLA* 0.20 High Fast Acid 70:30 27
Polymer 3 PLGA** 0.19 High Fast Acid 30:70 20
Polymer 4 PLGA** 0.19 Low Slow Ester 100:00; 70:30 20
Polymer 5 PLA* 0.32 Low Slow Ester 100:00; 70:30 27
* PLA = Poly (dl-lactide).
** PLGA = Poly (dl-lactide-co-glycolide).
a Comparative values expected on the basis of polymer structures.
b Maximum concentration obtained at 37 ◦ C.
 S. Chhabra et al. / International Journal of Pharmaceutics 342 (2007) 72–77 75

Fig. 4. In vitro percentage burst release of lysozyme from formulations 1–8

(F1–F8).

acterized by slower initial phase followed by faster second

phase. The second phase appeared at different time points (rang-
Fig. 2. Structure of polymers used, amino acid residues of lysozyme possess- ing from 2 to 12 weeks) for different formulations, which
ing OH group, and their interaction with polymer end groups. (a) Structure of could be explained on the basis of difference in polymer com-
polymer 1 (*OCH2 CH3 ), 2 (*OH), and 5 (*OCH2 CH3 ); (b) structure of poly- position, polymer end groups and/or composition of solvent
mer 3 (*OH) and 4 (*OCH2 CH3 ) where * and n represent the end group and
polymer chain length, respectively; (c) structure of threonine and serine amino
system.
acid residues having alcoholic hydroxyl groups; (d) possible interaction between Formulations 5 and 8 were made with polymers 2 and 3,
hydroxyl groups of amino acid residues of lysozyme and free carboxylic groups respectively, which had carboxylic acid as end groups, while
of polymers. other formulations made with polymers having ester as end
groups (Table 1 and Fig. 2a and b). Carboxylic acid end groups
posed of PLA only (Table 1 and Fig. 2a and b). Since, GA is might be thought to form some kind of chemical linkage (Fig. 2d)
relatively hydrophilic than PLA; the polymer 3 prefers a solvent with hydroxyl group bearing amino acid residues (Fig. 2c) of
system containing greater fraction of less hydrophobic solvent lysozyme, thereby, making the first phase of the biphasic release
component BA to get dissolved. pattern short and slow in comparison to those of rest of the
formulations. Therefore, the burst release determined for formu-
lations 5 and 8 were up to 4% while the rest of the formulations
3.2. In vitro release profile
showed burst release in the range of 20–30% of the incorporated
lysozyme (Fig. 4).
Fig. 3 shows the percentage cumulative release of lysozyme
The burst release is reported to be controlled by the rate
from formulations 1–8 at different time points through 20
of gelation. The higher the rate of gelation, greater would be
weeks. The general trend of release pattern is biphasic char-
the burst release and more hydrophobic (or less hydrophilic) a
formulation is, the sooner it would form the gel depot in situ
(Brodbeck et al., 1999a,b; Chen and Singh, 2005a,b; Singh and
Singh, 2004; Wang et al., 2003). Formulation 5 and 8 are rela-
tively less hydrophobic than other formulation; therefore, they
showed lower burst release which could be attributed to slower
rate of gelation expected with these formulations. The release in
first 24 h is treated as burst release.
After this initial burst release, the rest of the release is pre-
dominantly controlled by rate of degradation of polymer by
hydrolysis. The more hydrophobic a formulation is, the greater
it would resist the entry of water and therefore, the slower rate of
degradation. All of the formulations were prepared from poly-
mers of comparable molecular weight (IV 0.19–.22 dl/g) except
polymer 5 which is of greater molecular (IV 0.32 dl/g) and
has ester end group. Therefore, formulations 3 and 7 showed
significantly (p < 0.05) greater control on the release of incorpo-
rated lysozyme than rest of the formulation because they were
Fig. 3. In vitro percentage cummulative release of lysozyme from formulations prepared using polymer 5. Formulations 5 and 8 showed signif-
1–8 (F1–F8). icantly (p < 0.05) lower control over the release of incorporated
 76 S. Chhabra et al. / International Journal of Pharmaceutics 342 (2007) 72–77

Table 2
The sustained release formulations of lysozyme
Formulation Polymer Polymer concentration (w/v%) Lysozyme concentration (w/v%) Solvent %BB System %BA

Formulation 1 Polymer 1 15 5 100 0

Formulation 2 Polymer 4 15 5 100 0
Formulation 3 Polymer 5 15 5 100 0
Formulation 4 Polymer 1 15 5 70 30
Formulation 5 Polymer 2 15 5 70 30
Formulation 6 Polymer 4 15 5 70 30
Formulation 7 Polymer 5 15 5 70 30
Formulation 8 Polymer 3 15 5 30 70

lysozyme than other formulations because they were prepared 3.3. Biological activity of the released lysozyme
from polymers 2 and 3 containing carboxylic acid end groups,
respectively. Table 3 shows the MPSEA of lysozyme released from for-
Moreover, lysozyme was never released from formulations mulations 1–8. MPSEA was calculated for all formulations at
5 and 8 completely (maximum 92%) contrary to rest of the the time of exhaustion when no increase in cumulative per-
formulations where lysozyme got released up to 98% which centage release was found with time. The MPSEA values were
might be due to some kind of chemical linkages between car- found to be in the range of 30–52% which could be due to the
boxylic acid groups (available in formulations 5 and 8) and differences in exhaustion time. A formulation exhausting the
hydroxyl groups of lysozyme (Fig. 2d). Some of these link- release of lysozyme sooner, would be in contact with the releas-
ages might not have been hydrolyzed even after 10 or 12 weeks ing media for lesser amount of time; therefore, lesser decrease
which is supported by no increase in lysozyme in the released in its enzyme activity in comparison to formulation exhaust-
sample obtained after 10 weeks from formulation 5 and 8. Pos- ing in longer period of time. Therefore, formulation 8 showed
sible interaction of a peptide (leuprolide) with esterified end significantly greater (p < 0.05) MPSEA than other formula-
group of a polymer (PLGA) were implicated in explaining the tions as it got exhausted sooner (around 10 weeks) than other
release profile of leuprolide from a microsphere formulation formulations.
(Blanco and Alonso, 1998). In another study of chitosan based In 24-h samples lowest MPSEA was determined in samples
thermoset of indomethacin, its release at a controlled rate is from formulations 5 and 8 which might be due to the lower
reported to be due to the interaction of its aliphatic carbonyl amount of lysozyme released by these formulations. Formula-
group with NH2 group of the chitosan backbone (Gong et al., tion 5 and 8 are containing carboxylic acid end groups which
2006). have resulted in lower burst release from them.
Although formulations 3 and 7 are prepared from same Furthermore, a higher MPSEA observed in samples from for-
polymer 5, the rate of release of lysozyme from 7 was sig- mulation 5 and 8 indicates that the putative chemical linkage did
nificantly (p < 0.05) greater than that from 3 which might be not have any permanent destructive effect on the enzyme activ-
due to the difference in solvent system used in their prepa- ity of the lysozyme. This might be due to the regeneration of
ration (Table 2). Formulation 7 used a mixture of BB and hydroxyl groups involved in the possible covalent bond forma-
BA (70:30) whereas only BB was used for formulation 3. BA tion with the carboxylic group via the mechanism of hydrolysis.
being more hydrophilic than BB, comes out sooner during the In all the formulations, MPSEA was found to be greater in com-
gelation process resulting in relatively increased porosity of parison to MPSEA observed in lysozyme solution kept under
the gel depot formed. Porosity facilitates access of water to similar conditions (37 ◦ C at 35 rpm) at all the time points except
the polymer back-bone which causes polymer degradation via day 1. The decrease in MPSEA in formulation on day 1 might be
the hydrolysis process (Graham et al., 1999; Eliaz and Kost, due to influence of solvent on surrounding media on lysozyme.
2005). However, a better MPSEA in formulations at all other time points

Table 3
Percentage specific enzyme activity (%SEA)a (mean ± S.D; n = 3) of lysozyme released from various formulations
Weeks Lysozyme F1 F2 F3 F4 F5 F6 F7 F8

0 100 ± 0.0 ND ND ND ND ND ND ND ND
0.143 97 ± 1.5 94 ± 0.6 96 ± 0.6 95 ± 0.6 96 ± 0.9 85 ± 2.5 97 ± 0.4 96 ± 1.1 87 ± 2.7
4 64 ± 2.1 70 ± 0.8 85 ± 0.8 65 ± 1.2 76 ± 0.8 65 ± 0.7 87 ± 0.8 71 ± 0.9 67 ± 1.8
8 49 ± 1.8 59 ± 0.5 64 ± 1.1 52 ± 0.5 61 ± 1.4 55 ± 1.9 66 ± 0.6 56 ± 0.9 56 ± 1.5
12 26 ± 0.8 45 ± 1.8 51 ± 0.5 38 ± 1.7 48 ± 1.7 49 ± 1.8 50 ± 0.9 41 ± 0.6 52 ± 1.1
16 10 ± 0.7 32 ± 0.7 35 ± 2.1 35 ± 2.1 30 ± 2.1 35 ± 1.2 ND ND ND
20 ND ND ND ND 22 ± 1.9 ND ND ND ND

F, formulation; ND, not detected.

a SEA (%) = SEA in sample
SEA in freshly prepared lysozyme × 100.
 S. Chhabra et al. / International Journal of Pharmaceutics 342 (2007) 72–77 77

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