Powder Technology 205 (2011) 65–70

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Powder Technology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p ow t e c

Production and characterization of chitosan microparticles containing papain for

controlled release applications
Fernando C. Vasconcellos, Gilberto A.S. Goulart, Marisa M. Beppu ⁎
School of Chemical Engineering/ State University of Campinas (UNICAMP), Cidade Universitária "Zeferino Vaz", CEP 13083-852 - Campinas - SP - Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The production and modification of chitosan microparticles using crosslinking agents and papain were
Received 1 October 2009 evaluated for controlled release applications. Chitosan microparticles were produced and crosslinked with
Received in revised form 13 July 2010 sodium tripolyphosphate (TPP) 10% (w/v) solution or glutaraldehyde (GLU) 0.75% (w/w), with subsequent
Accepted 30 August 2010
papain sorption. Microparticles were characterized by Fourier transformed infrared spectroscopy (FTIR) for
Available online 16 September 2010
chemical modifications, scanning electron microscopy (SEM) for morphology and X-ray diffraction (XRD) for
crystallographic analysis. Chemical composition and the thermal stability of the material were characterized
Keywords:
Chitosan
by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). It was observed that the
Microparticles presence of TPP and papain resulted in a decrease of the stability of the chitosan matrix. Papain release rates
Papain from the microparticles were also conducted in vitro. The amount of released papain in phosphate buffer
Controlled release (pH = 7.4) was analyzed with UV-spectroscopy, showing release profiles of enzymatic activity ranging from
Biomaterial 0.006 to 0.011 μmol.min− 1. The results indicate that both chitosan–TPP–papain and chitosan–GLU–papain
microparticles can successfully be used for systems that aim at a controlled release of papain with potential
use in the biomedical area.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction applications due to better packing, in comparison to monodisperse

Interest in biomedical applications of biopolymers is growing
quickly mainly due to their remarkable biocompatibility and
biodegradability properties, among many others. Chitosan — a
polysaccharide derived from chitin, obtained mainly by extraction
from crustacean shells such as crab and shrimp, is such an example. It
has been studied for many medical applications such as tissue
engineering and drug delivery systems [1,2]. When it is placed in
contact with biological tissues, it does not provoke immunogenic
reactions, while it promotes cellular adhesion and inhibits scar
formation [3]. Another advantage of using chitosan in tissue
regeneration is that it allows optimum oxygen permeability for all
the tissues and avoids the loss of body fluids [4].
Depending on its final application, chitosan can be prepared as
powders [5,6], membranes [4,7], gels [8,9] or spherical microparticles
[10–14] and nanoparticles [14,15]. Chitosan microparticles are widely
used in drug release applications. Different drug–chitosan combina-
tions allow it to be tailored for specific treatments in different sites:
small sizes, for example, induce large surface to volume ratios and can
be used in parallel for controlled release of insoluble drugs [16], while
polydisperse systems are particularly suitable for wound healing
⁎ Corresponding author. Tel.: + 55 19 3521 3900; fax: + 55 19 3521 3910.
E-mail address: beppu@feq.unicamp.br (M.M. Beppu).
systems.
Chitosan has been used previously to obtain microparticles and
nanoparticles by solvent evaporation, coacervation, and spraying
methods. There are several advantages in using the spraying method:
(1) when water is used as a solvent there are no toxic solvents, oils, or
emulsifiers that may leave undesirable residuals in the final
microparticle formulation; (2) the spraying method may achieve
high productivity and is easily scaled-up; (3) the method is suitable
for wound healing applications due to the production of polydisperse
systems of chitosan microparticles as mentioned above.
Papain is a proteolytic enzyme extracted from Carica papaya,
which can be used to increase chitosan's wound healing capability.
Papain presents anti-inflammatory, antibacterial, and antioxidant
properties [17–19] and can be used in the treatment of large skin
lesions. This is attributed to its ability to remove injured tissues and to
stimulate the healing process. In tissue regeneration, this natural
product is also useful in reducing the bacterial burden, decreasing
exudates, and increasing granulation tissue formation [20]. Commer-
cially, papain has been used as an enzymatic debridement agent
available in products for wound treatment [21,22].
Powder agents can be more successful than films in wound healing
processes of areas that require flexibility and that do not need
mechanical immobilization. The combination of chitosan microparti-
cles and papain has the potential to provide a successful wound
dresser for skin damage recovery and has not been reported yet.
However, as papain can cause chitosan depolymerization, either

0032-5910/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.powtec.2010.08.066
66 F.C. Vasconcellos et al. / Powder Technology 205 (2011) 65–70
chitosan must be present in a crosslinked form or papain needs to be
immobilized in the polymeric matrix [23,24].
Chemical crosslinking agents such as glutaraldehyde, ethylene
glycol diglycidyl ether and poly(ethylene glycol), have been used to
enhance controlled release of drugs from chitosan microparticles [25].
The addition of these substances might be limited due to their toxicity.
An alternative is to use a less toxic crosslinking agent such as
tripolyphosphate (TPP). It acts by increasing the pH and ionic strength
of the solution, forming a gel and promoting ionic interaction between
the amino groups of chitosan and the anionic groups of TPP [26].
Studies have shown that chitosan microparticles produced by ionic
crosslinking with TPP increased the drug loading efficiency in the
chitosan microparticles and also prolonged the drug release period
[27,28].
The objective of this study was to evaluate the preparation and
modification of chitosan microparticles using crosslinking agents, and
to study the papain release rate from these microparticles, aiming at
controlled-release applications.
2. Experimental
2.1. Materials
Chitosan was obtained from Sigma-Aldrich (USA) with a minimum
deacetylation degree of approximately 75%. Tripolyphosphate from
Synth® (Brazil) and glutaraldehyde from Nuclear® (Brazil) were used
as crosslinking agents. Papain (Brauzyn100) was kindly provided by
the School of Pharmaceutical Sciences at the State University of São
Paulo (USP-Brazil). All other reagents were of analytical grade, and
used without further purification. All solutions were prepared with
deionized ultrapure Milli-Q® (18 MΩ) water.
2.2. Methods
2.2.1. Preparation of microparticles
Chitosan microparticles were prepared from a solution of 1.0% w/
w in acetic acid (3% v/v), sprayed through a nozzle into a reacting tank
containing a coagulant 1 M NaOH solution. A schematic representa-
tion of the system used to produce the chitosan microparticles, Spray
Systems do Brasil (1/4JN-SS + SU11-SS), is shown in Fig. 1.
Chitosan solution flow rate was kept constant at 9 mL.min−1 and
the nitrogen flow was controlled at a pressure of 2.25 kgf.cm−2. After
spraying, chitosan microparticles were kept in the NaOH solution for
24 h at room temperature to complete coagulation. The particles were
separated from the coagulant solution by filtering with GRANUTEST
sieves with 53 μm pore sizes. The microparticles were extensively
rinsed with Milli-Q® (18MΩ) water to remove NaOH until neutral pH
Fig. 1. Schematic representation of the system used to produce the chitosan
microparticles.
was achieved. Finally, the microparticles were stored in Milli-Q®
water at 4 °C.
2.2.2. Chemical modification of the microparticles
Chitosan microparticles were heterogeneously crosslinked by
immersion in either a 0.75% w/w aqueous glutaraldehyde (GLU)
solution or a 10% w/v tripolyphosphate (TPP) solution, for 2 h at room
temperature, followed by rinsing with Milli-Q® water to remove the
unreacted crosslinking residues.
2.2.3. Papain sorption
Pristine and crosslinked chitosan microparticles were immersed in
a 1% w/v papain solution for 12 h to promote papain sorption. The
particles were separated by filtration from the solution and washed
with Milli-Q® water, then immersed in liquid nitrogen (−195 °C) and
lyophilized, at a temperature of −47 °C during 24 h.
2.2.4. In vitro papain release studies
For the in vitro papain release studies, microparticles (10 mg)
containing papain were suspended in 40 mL of phosphate-
buffered saline (PBS, pH 7.4) solution, at 37 °C. The solution was
agitated at a frequency of 100 rpm during a 24 h period. At
predetermined 1 h time intervals, 2 mL samples of the supernatant
were withdrawn. To determine the amount of papain released, the
following were added to the 2 mL sample: 2 mL EDTA (1 mM), 2 mL
cysteine (5 mM), 2 mL casein (1%) and 2 mL phosphate-buffer (pH
7.4). The mixture was left to rest for 30 min, followed by the addition
of trichloroacetic acid (6 mL, 5% w/v) to the solution to stop the
enzymatic reaction. After 15 min of centrifugation, the amount of
papain released was determined by the amount of tyrosine produced
in the reaction with casein through UV-spectroscopy peak measure-
ments at λ = 280 nm. These measurements were performed in
triplicate.
2.3. Microparticle characterization
Chitosan microparticle distribution size was determined by
dynamic light scattering (Mastersizer S, Malvern) in the 10 μm to
1000 μm range, before the microparticles were filtered through 53 μm
pore sieves, crosslinked and loaded with papain.
The chitosan microparticles were freeze dried and analyzed with
scanning electron microscopy (SEM). Samples were coated with a thin
gold layer (~ 10 nm) using a sputter coater SCD 050 (Baltec,
Liechenstein) and observed on a JEOL JXA-840 field emission scanning
electron microscope (25 kV) (Jeol, Japan).
The crystallinity of pristine chitosan powder, pure chitosan
microparticles, and chitosan microparticles crosslinked with TPP
were determined by X-ray diffractometry (X'Pert PW3050 Philips),
with a scanning scope of 5–60°, scanning speed of 8°/min, and Cu-Kα
radiation (λ = 1.5406.10−10 m). The spectra were obtained utilizing
PC-APD software version 4.0, and MS origin 5.0.
The spectroscopic structural study of the chitosan microparticles
was performed by Fourier transform infrared spectroscopy (FTIR-
ATR) utilizing a Nicolet Protegé 460. Spectral scanning was acquired
in the 2000 to 500 cm−1 range.
Thermogravimetric analysis (TGA) and differential scanning
calorimetry (DSC) studies were performed on the pure chitosan
microparticles and on chitosan microparticles crosslinked with TPP.
TGA was performed using a TGA-50 (Shimadzu) with a heating ramp-
up rate of 10 °C.min− 1, in a nitrogen atmosphere with a flow rate of
25 mL.min− 1. DSC analysis was performed with a DSC-50 (Shimadzu)
analyzer. Samples (approx. 5–7 mg) were scanned in sealed platinum
(6 × 1.5 × 1.5 mm) trays and heated up to a temperature of 450 °C at a
rate of 10 °C.min− 1, under a nitrogen atmosphere having a flow rate
of 50 mL.min− 1.
 F.C. Vasconcellos et al. / Powder Technology 205 (2011) 65–70 67

final size distribution of 10–53 μm diameter microparticles. For

0.08 wound healing applications, a polydisperse system, as the one
obtained, is desirable due to its ability for improved particle packing,
Tyrosine concentration (mM)

in comparison to monodisperse systems.

SEM images of freeze-dried chitosan microparticles crosslinked with
0.06
TPP and GLU can be seen in Fig. 4. Chitosan–GLU microparticles
presented a microporous surface with no homogeneity in shape. The
crosslinking with GLU favored microporous formation differently from
0.04 the non-crosslinked chitosan particle structures. The chitosan–GLU
microparticles presented a smoother surface morphology (Fig. 4b) than
that of the non-crosslinked chitosan microparticles (Fig. 4a) — a result
corroborated by other authors [28]. The heterogeneity of shape may be a
0.02

0
0 1 2 3 4 5 6 7
Days

Fig. 2. Enzymatic activity of papain at 37 °C, measured by tyrosine concentration (mM)

as a function of time.

3. Results and discussion

3.1. Enzymatic activity of papain

Enzymatic tests were performed to verify the stability of the

enzyme for a period of 7 days at a temperature of 37 °C. Fig. 2 shows
that the enzymatic activity was kept constant during the entire period
of the test. Had the degradation of papain occurred, it would lead to
errors in the quantification method of the controlled release after
encapsulation, which was not the case in our study.

3.2. Characterization of the microparticles

By analyzing the chitosan microparticles after preparation, a

bimodal behavior was observed for the particle size distribution
(Fig. 3). The microparticle sizes ranged from 10 to 800 μm in diameter,
with bimodal peaks at approximately 70 and 400 μm diameters. This
can be explained by the difficulty of the microparticles to retain their
shape when in contact with the coagulation solution due to the low
viscosity of the 1% chitosan solution, and also due to the tendency of
aggregation of the particles to occur after coagulation, which could be
observed by SEM images. The Sauter mean diameter for the chitosan
microparticles was calculated to be 121 μm. Before further processing,
these microparticles were filtered through 53 μm sieves, obtaining the

Fig. 3. Pure chitosan microparticles distribution as a function of their respective Fig. 4. SEM images of (a) pristine chitosan, (b) chitosan–GLU and (c) chitosan–TPP
diameters, analyzed with dynamic light scattering (laser diffraction). crosslinked microparticles (scale bars = 20, 25 and 25 μm, respectively).
68 F.C. Vasconcellos et al. / Powder Technology 205 (2011) 65–70
Fig. 5. FTIR-ATR spectra of chitosan microparticles after freeze-drying of: (a) pristine
chitosan, (b) chitosan–TPP–papain and (c) chitosan–GLU–papain microparticles.
result of the drying process, observed both for crosslinked and non-
crosslinked chitosan particles. Chitosan–TPP microparticles (Fig. 4c)
showed macropore surfaces with follicular structure appearance.
The morphology of neither the chitosan–GLU and chitosan–TPP
microparticles was the deciding factor to opt for the best crosslinking
agent. Due to its lower toxicity, TPP was chosen as the primary
crosslinking agent.
FTIR-ATR spectra of pristine chitosan, chitosan–TPP and chitosan–
GLU microparticles are depicted in Fig. 5. Fig. 5a shows the spectrum
obtained for pristine chitosan microparticles where the peaks for the
aliphatic groups, charged amino groups, and carboxylic acid groups are
at 1100 cm− 1, 1500 cm−1, and 1450 cm− 1, respectively. The charged
amino and carboxylic acid group peaks showed slightly shifted positions
Fig. 6. X-ray diffraction patterns of (a) pure chitosan powder, (b) pristine chitosan microparticles, (c) chitosan microparticles with papain and (d) crosslinked chitosan–TPP
microparticles with papain.
with respect to the peaks obtained from pristine chitosan membranes
reported in the literature [29] that appear at 1100 cm− 1 for aliphatic
amines; 1550 cm− 1 for charged amino groups; and 1400 cm− 1
for carboxylic acid (COO−) groups. The spectrum obtained for
chitosan–TPP–papain microparticles (Fig. 5b) shows two new peaks at
1150 cm− 1 and 1200 cm− 1, both corresponding to -P= O groups,
indicating the presence of TPP. Chitosan–GLU microparticles present the
following typical peaks at: 1100 cm− 1 for aliphatic groups from
glutaraldehyde; 1550 cm− 1 for - N = C groups; 1560 cm− 1 for -C= C
groups; and 1720 cm− 1 for free aldehyde groups.
X-ray diffraction patterns shown in Fig. 6 correspond to pure
chitosan powder, and lyophilized chitosan, chitosan–papain, and
chitosan–TPP–papain microparticles. The diffractogram of pure
chitosan powder (Fig. 6a) shows two distinct peaks for the scattering
angle 2θ, equal to 10.4o and 22o approximately. Lyophilized pristine
chitosan microparticles (Fig. 6b) showed a modified scattering
pattern in comparison to the chitosan powder, with four diffraction
peaks corresponding to a scattering angle 2θ, equal to 20o, 22o, 32o
and 33o approximately. The diffractogram for the chitosan–papain
microparticles (Fig. 6c) indicated that these microparticles became
more amorphous in comparison to the pristine chitosan microparti-
cles. Only one diffraction peak is noticeable, at 2θ equal to 22o. The loss
in degree of crystallinity is probably due to the non-uniform insertion
of papain within the chitosan polymer chains of the microparticle.
Chitosan particles crosslinked with TPP containing papain showed a
more crystalline diffractogram, with the appearance of additional
diffraction peaks, in comparison to the chitosan–papain microparti-
cles, as seen in Fig. 6d. This result is expected due to the higher
organization that the TPP crosslinking confers to the chitosan polymer
chains. The non-uniformity of the inter-planar distance between the
chitosan chains, along with the insertion of papain in different
sections of the crosslinked polymer matrix, lead to the appearance of
 F.C. Vasconcellos et al. / Powder Technology 205 (2011) 65–70
Fig. 7. TGA thermograms of freeze-dried (a) pristine chitosan and (b) chitosan–TPP–
papain microparticles.
Fig. 8. DSC results for freeze-dried (a) pristine chitosan and (b) chitosan–TPP–papain
microparticles.
Fig. 9. Release rate profile of papain (enzymatic activity (μmol/min) as a function of time) for (a) chitosan–TPP–papain, and (b) chitosan–GLU–papain microparticles, during 25 h.
69
additional, and at times, overlapping peaks. It is expected that papain
will diffuse at a faster rate in microparticles that are amorphous due to
pore formation and larger exchange surface area, in comparison to the
more crystalline structured chitosan–TPP microparticles. Therefore,
crosslinked particles are more suitable for controlled release applica-
tions that require slow release rates.
The studies on the thermal behavior of the chitosan micropar-
ticles by TGA are shown in Fig. 7. Freeze-dried pristine chitosan
and chitosan–TPP–papain microparticles showed similar thermal
behavior. However, TPP-modified microparticles with papain, curve
(b), have a slightly reduced thermal resistance in the 0 °C to 100 °C
and 270 °C to 570 °C ranges, in comparison to that of pristine chitosan
microparticles without papain, curve (a). Studies have shown that
crosslinking chitosan with TPP results in an increase in hydrophilicity
of the polymer matrix [30] and therefore in higher water retention,
what explains the observation of the first reduction in thermal
resistance in the 0 °C to 100 °C range, where TPP-crosslinked
microparticles loose greater amounts of water in comparison to
non-crosslinked particles that retain less water.
Fig. 8 shows the DSC results for the pristine chitosan and the
chitosan–TPP–papain microparticles in curves (a) and (b), respec-
tively. For the pristine chitosan microparticles, the endothermic valley
observed at 100 °C corresponds to the loss of water by these
microparticles. Endothermic peaks are observed below 100 °C for
the chitosan–TPP crosslinked microparticles, probably due to the
presence of ionic TPP. Since TPP is hydroscopic, it will retain water
contained in the chitosan–TPP microparticle for a longer period of
time as the temperature is gradually increased. The three endother-
mic peaks between 0 °C and 150 °C can probably be attributed to
separate stages of water removal. The curve for the chitosan–TPP
crosslinked microparticles presented exothermic peaks at tempera-
tures of approximately 165 °C and 295 °C, which are lower in
comparison to those of pristine chitosan microparticles at 170 °C
and 305 °C, approximately. This is an indication of the degradation of
papain present only in the chitosan–TPP–papain microparticles, fact
that was also observed in the TGA analysis.
70 F.C. Vasconcellos et al. / Powder Technology 205 (2011) 65–70
3.2. In vitro release of papain from chitosan microparticles
Tests of the in vitro release of papain from the chitosan
microparticles were performed in order to evaluate release char-
acteristics of papain from chitosan microparticles crosslinked with
TPP and GLU. The amount of papain sorbed in the chitosan–GLU and
chitosan–TPP microparticles, in terms of (g of papain/g of micro-
particles), was determined to be 0.03 g and 0.04 g, respectively,
measured using the spectrophotometer to quantify papain activity on
the casein substrate. The in vitro release tests were performed in
triplicate, and the average is presented in Fig. 9. The results show that
there was no significant difference in the release rate of papain from
chitosan microparticles crosslinked with TPP versus microparticles
crosslinked with GLU. The papain release rates for both microparticles
showed a slightly increasing profile, indicating that papain is released
in similar proportions during the first 25 h.. The measured enzymatic
activity ranged from 0.006 to 0.011 μmol.min− 1. These profiles also
indicate that the release rate of papain is probably limited by mass
transfer effects of papain present in the microparticles, and not by
how papain is incorporated in the chitosan microparticles.
4. Conclusion
The use of TPP and GLU as crosslinking agents revealed to be viable
for chitosan microparticles. Papain release profiles from the micro-
particles with TPP and GLU in phosphate buffer (pH 7.4) showed
similar, slightly increasing release profiles. These results show that
both chitosan–GLU–papain and chitosan–TPP–papain microparticles
are strong candidates for the control release application of papain. In
face of these results, TPP should preferably be used as a crosslinking
agent in this application due to its lower toxicity in comparison with
GLU. In conclusion, the results obtained for in vitro release tests are
very promising, regarding its use in the pharmaceutical area. Future
experiments in wound healing shall be done to confirm this potential.
Acknowledgements
The authors gratefully acknowledge the financial support from
CAPES, CNPq and FAEPEX-UNICAMP.
References
[1] M.D. Blanco, C. Gómez, R. Olmo, E. Muñiz, J.M. Teijón, Chitosan microspheres in
PLG films as devices for cytarabine release, Int. J. Pharm. 202 (2000) 29–39.
[2] E. De Souza Costa Jr, M.M. Pereira, H.S. Mansur, Properties and biocompatibility of
chitosan films modified by blending with PVA and chemically crosslinked, J.
Mater. Sci. Mater. Med. 20 (2009) 553–561.
[3] Q. He, T. Zhang, Y. Yang, F. Ding, In vitro biocompatibility of chitosan-based
materials to primary culture of hippocampal neurons, J. Mater. Sci. Mater. Med. 20
(2009) 1457–1466.
[4] G.G. Aimoli, M.A. Torres, M.M. Beppu, Investigations into the early stages of “in
vitro” calcification on chitosan films, Mat. Sci. Eng. C 26 (2006) 78–86.
[5] T.P. Learoyd, J.L. Burrows, E. French, P.C. Seville, Modified release of beclometa-
sone dipropionate from chitosan-based spray-dried respirable powders, Powder
Technol. 187 (2008) 231–238.
[6] R.J. Garmise, K. Mar, T.M. Crowder, C.R. Hwang, M. Ferriter, J. Huang, J.A. Mikszta,
V.J. Sullivan, A.J. Hickey, Formulation of a dry powder influenza vaccine for nasal
delivery, AAPS PharmSciTech 7 (2006) E1–E7.
[7] G. Cárdenas, P. Anaya, C. Von Plessing, C. Rojas, J. Sepúlveda, Chitosan composite
films. Biomedical applications, J. Mater. Sci. Mater. Med. 19 (2008) 2397–2405.
[8] S. Kumar, J. Dutta, P. K. Dutta, Preparation and characterization of N-heterocyclic
chitosan derivative based gels for biomedical applications. Int. J. Biol. Macromol.,
Article in Press. DOI: 10.1016/j.ijbiomac.2009.08.002.
[9] C.Z. Wei, C.L. Hou, Q.S. Gu, L.X. Jiang, B. Zhu, A.L. Sheng, A thermosensitive
chitosan-based hydrogel barrier for post-operative adhesions' prevention,
Biomaterials 30 (2009) 5534–5540.
[10] F.L. Mi, H.W. Sung, S.S. Shyu, Release of indomethacin from a novel chitosan
microsphere prepared by a naturally occurring crosslinker: examination of
crosslinking and polycation-anionic drug interaction, J. Appl. Polym. Sci. 81
(2001) 1700–1711.
[11] J.A. Ko, H.J. Park, S.J. Hwang, J.B. Park, J.S. Lee, Preparation and characterization of
chitosan microparticles intended for controlled drug delivery, Int. J. Pharm. 249
(2002) 165–174.
[12] V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R. Kumria, K. Bansal, S. Dhawan,
Chitosan microspheres as a potential carrier for drugs, Int. J. Pharm. 274 (2004)
1–33.
[13] P.B. Malafaya, A.J. Pedro, A. Peterbauer, C. Gabriel, H. Redl, R.L. Reis, Chitosan
particles agglomerated scaffolds for cartilage and osteochondral tissue engineer-
ing approaches with adipose tissue derived stem cells, J. Mater. Sci. Mater. Med. 16
(2005) 1077–1085.
[14] B. Combadière, B. Mahé, Particle-based vaccines for transcutaneous vaccination,
Comp. Immunol. Microbiol. Infect. Dis. 31 (2008) 293–315.
[15] L.B. Peppas, B. Ghosn, K. Roy, K. Cornetta, Encapsulation of nucleic acids and
opportunities for cancer treatment, Pharm. Res. 24 (2007) 618–627.
[16] S. Vasiliu, M. Popa, C. Luca, Evaluation of retention and release processes of two
antibiotics from the biocompatible core-shell microparticles, Eur. Polym. J. 44
(2008) 3894–3898.
[17] A. Emeruwa, Antibacterial substance from Carica papaya fruit extract, J. Nat. Prod.
45 (1982) 123–127.
[18] G. Rimbach, Q. Guo, T. Akiyama, S. Matsugo, H. Moini, F. Virgili, L. Packer, Ferric
nitrilotriacetate induced DNA and protein damage: inhibitory effect of a
fermented papaya preparation, Anticancer Res. 20 (2000) 2907–2914.
[19] O.P. Gupta, S. Sing, S. Bani, N. Sharma, S. Malhotra, B.D. Gupta, S.K. Banerjee, S.S.
Handa, Anti-inflammatory and anti-arthritic activities of silymarin acting through
inhibition of 5-lipoxygenase, Phytomedicine 7 (2000) 21–24.
[20] D. Telgenhoff, K. Lam, S. Ramsay, V. Vasquez, K. Villareal, P. Slusarewicz, P. Attar, B.
Shroot, Influence of papain urea copper chlorophyllin on wound matrix
remodeling, Wound Repair Regen. 15 (2007) 727–735.
[21] P.A. Hebda, C.-Y. Lo, Evaluation of the efficacy of enzymatic debriding agents for
removal of necrotic tissue and promotion of healing in porcine skin wounds,
Wounds 13 (2001) 190–194.
[22] A.F. Falabella, Debridement and wound bed preparation, Dermatol. Ther. 19
(2006) 317–325.
[23] R.A.A. Muzzarelli, P. Ilari, R. Tarsi, B. Dubini, W. Xia, Chitosan from Absidia coerulea,
Carbohydr. Polym. 25 (1994) 45–50.
[24] L.Y. Lim, L.S.C. Wan, P.Y. Thai, Chitosan microspheres prepared by emulsification
and ionotropic gelation, Drug Dev. Ind. Pharm. 23 (1997) 981–985.
[25] X.Z. Shu, K.J. Zhu, A novel approach to prepare tripolyphosphate/chitosan
complex beads for controlled release drug delivery, Int. J. Pharm. 201 (2000)
51–58.
[26] F.L. Mi, S.S. Shyu, C.Y. Kuan, S.T. Lee, K.T. Lu, S.F. Jang, Chitosan-polyelectrolyte
complexation for the preparation of gel beads and controlled release of anticancer
drug. I. Effect of phosphorus polyelectrolyte complex and enzymatic hydrolysis of
polymer, J. Polym. Sci. 74 (1999) 1868–1879.
[27] X. Shu, K. Zhu, Preprints of 2nd International Symposium on High-tech Polymers
and Polymeric Complex (HPPC-II), , 1999.
[28] K.C. Gupta, F.H. Jabrail, Glutaraldehyde cross-linked chitosan microspheres for
controlled release of centchroman, Carbohydrate Res. 342 (2007) 2244–2252.
[29] M.M. Beppu, C.C. Santana, PAA influence on chitosan membrane calcification,
Mater. Sci. Eng. C 23 (2003) 651–658.
[30] D. R. Bhumkar, V. B. Pokharkar, Studies on Effect of pH on Cross-linking of
Chitosan With Sodium Tripolyphosphate: A Technical Note. AAPS PharmSciTech.
2006; 7(2): Article 50.