Food Hydrocolloids 18 (2004) 865–871

www.elsevier.com/locate/foodhyd

Formation and stability of shark liver oil loaded

chitosan/calcium alginate capsules
C. Penichea, I. Howlandb, O. Carrilloc, C. Zaldı́varc, W. Argüelles-Monald,*
a
Centro de Biomateriales, Universidad de La Habana, La Habana 10400, Cuba
b
Centro de Investigaciones Médico Quirúrgicas (CIMEQ), La Habana, Cuba
c
Facultad de Biologı́a, Universidad de La Habana, La Habana 10400, Cuba
d
CIAD-Unidad Guaymas, Carretera al Varadero Nacional Km 6.6, Apdo. Postal 284, Guaymas, Sonora, 85480 Mexico

Abstract
Calcium alginate capsules containing shark liver oil were prepared by ionotropic gelation of alginate solutions. Since the capsules were
permeable to the oil, they were coated with a membrane of chitosan-alginate polyelectrolyte complex to reduce permeability. Encapsulation
efficiencies (expressed as the percentage of shark liver oil entrapped) greater than 87% (w/w) were obtained by using 6% (w/v) alginate
solution. The oil content in the capsules was higher than 65% (w/w) when the oil was dispersed in the alginate solution at 10% (v/v) or more.
However, at the level of 15 or 20% (v/v) the oil was exuded after 48 h. Capsules were degraded in vitro by enzymes, with lipase being more
effective than pancreatine. No release of oil from the capsules up to 4 h at pH 1.2 was observed, but after 4 h the capsules became very fragile
when they were immersed in buffer solution at pH 7.4.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: Encapsulation; Shark liver oil; Chitosan; Alginate

1. Introduction It has been well established that the guluronic units are
the ones responsible for the crosslinking reaction and
There has been growing interest in the development of therefore the properties of the beads formed such as
encapsulation procedures for applications in biotechnology, strength and porosity will depend on the alginate source.
biomedicine, and pharmacy (Chang, 1992; Sun, Vacek, & Other parameters that can have an effect on the
Tai, 1992). Capsules can be made from polyelectrolyte characteristics of beads are the alginate molecular weight,
complexes through the mixing of oppositely charged and the concentration of CaCl2 and alginate solution (Thu
macromolecules. Although many combinations of polya- et al., 1996a).
nion – polycation can be used to form capsules, a widely used If the alginate solution containing a substance dissolved
system is to dispense a sodium alginate solution containing or suspended into it is dropped into the calcium chloride
the material to be encapsulated into a calcium chloride solution, the substance becomes encapsulated as the result
solution to form calcium alginate beads. These beads are then of ionotropic gelation (Vorlop & Klein, 1981).
immersed in solutions of poly-L -lysine or chitosan (Thu et al., The mechanical properties and permeability of sodium
1996b; Yao, Peng, Yin, Xu, & Goosen, 1996). alginate capsules can be modified by placing the capsules
Alginate is the name given to a family of linear into a solution of a polycation, such as chitosan. Chitosan
polysaccharides found in brown algae composed by
is a polysaccharide composed essentially by b(1 – 4)
guluronic (G) and manuronic (M) units. The M/G ratio
linked gluosamine units together with some proportion of
and their distribution along the chains (microstructure) are
N-acetylglucosamine units. It is obtained by extensive
strongly dependent on the particular species of algae from
deacetylation of chitin, a polysaccharide widely spread in
which it was extracted (Thu et al., 1996a).
nature (Gåserød, Sannes, & Skjåk-Bræk, 1999).
Calcium alginate beads are produced by ionotropic
Chitosan reacts with alginate to form a polyelectrolyte
gelation of alginate in the presence of calcium ions as
shown in Scheme 1. complex as shown in Scheme 2.
The extent of this reaction is strongly pH-dependent. As
* Corresponding author. Tel. and Fax: þ52-622-221-65-33. a result of this reaction, the alginate beads become covered
E-mail address: [email protected] (W. Argüelles-Monal). by a chitosan shell. The thickness of this layer depends on
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2004.02.001
866 C. Peniche et al. / Food Hydrocolloids 18 (2004) 865–871

(427 U/L at 37 8C) was obtained from Boehringer Man-

nheim (Germany). Shark liver oil was kindly supplied as a
Scheme 1.
crude extract by the Centro de Investigaciones Pesqueras
(Havana, Cuba).

Scheme 2.
2.1. Purification of sodium alginate
the molecular weight of chitosan as well as on the pH and
chitosan concentration of the solution. A 0.5% (w/v) solution of sodium alginate was succes-
Of all the factors affecting the properties of the capsules, sively filtered through glass – wool and 120, 90 and 45 mm
in the present work we focused our attention on the pore-sized sintered glass filters. NaCl was slowly added to
influence of sodium alginate concentration, the amount of the filtrate with stirring until the salt concentration reached
oil to be encapsulated and the effect of chitosan cover on the 1 mol/L. Then absolute ethanol was added with agitation
permeability of the capsules and on the encapsulation until the alcohol concentration in the mixture was 70%
efficiency (EE). (v/v). The precipitated polymer was separated from the
Shark liver oil is a good source of nutritional factors such alcohol solution by filtration through a 45 mm pore-sized
as lipid soluble vitamins and essential fatty acids. Of great sintered glass filter. The solid filter cake collected on the
interest is its composition rich in v-3 polyunsaturated fatty filter surface was washed successively with 80, 90 and 95%
acids (PUFA). It has been confirmed that these v-3 PUFA (v/v) ethanol solutions and finally with absolute ethanol.
retard the formation of clots responsible for cardiovascular The resultant purified sodium alginate was dried in vacuum
diseases. There is a close relationship between the consump- at room temperature for 48 h.
tion of the oil and the decrease of cholesterol and triglycerids
level in blood, together with a decrease in blood pressure,
factors that reduce the risk of suffering coronary atheroma- 2.2. Encapsulation procedure
tosis (Goodnight, Fisher, Fitzgerald, & Levine, 1989).
However, shark liver oil has unpleasant taste and odour, A specific amount of shark liver oil was mixed with
which constitutes a serious inconvenience for its use as a 10 ml of an aqueous solution of sodium alginate in a 50 ml
dietary supplement or therapeutic use in humans as well as beaker and stirred using a mechanical stirrer at 800 rpm for
for the nourishment of marine animal larvae of economic 15 min at room temperature in order to form an oil-in-water
interest. Moreover, due to its high content of polyunsatu- emulsion. Sodium alginate concentration and oil:alginate
rated fatty acids shark liver oil easily undergoes oxidation solution ratios used are reported in Table 1. The emulsion
when exposed to air or UV radiation. To overcome these was dropped with a syringe in a beaker containing 10 ml of
drawbacks, we have microencapsulated the oil in calcium 2% (w/v) CaCl2 solution under gentle stirring. The capsules
alginate beads coated with chitosan. The influence of formed were agitated for 10 min, decanted and the
different parameters of the capsule preparation process on supernatant was discarded. Afterwards 10 ml of 1% (w/v)
their characteristics as well as the behaviour of the capsule’s chitosan solution in 0.1 M HCl solution was added while
membrane under the action of digestive enzymes and stirring for another 10 min. In order to remove the
changes in the pH of the medium was studied. unbounded chitosan at the surface, the capsules were
immersed in 10 ml of a 1% (w/v) CaCl2 solution and
agitated for another 10 min. CaCl2 solution was used instead
2. Materials and methods of water for avoiding any lost of calcium. The capsules
obtained were washed with absolute ethanol and dried at
Chitosan was used as supplied by Protan (Biopolymer, room temperature. The average diameter of the capsules
Drammen, Norway). Its viscosity – average molecular ranged from 1.4 to 2 mm.
weight was Mv ¼ 6:9 £ 105 as estimated at 25 8C in 0.3 M
Table 1
acetic acid/0.2 M sodium acetate (Rinaudo, Milas, & Le Concentration of alginate solution, percentage of oil suspended, EE and oil
Dung, 1993) and the degree of N-acetylation, DA ¼ 0.18 content of capsules (OC)
was determined by 1H NMR.
Experiment Alginate Shark liver oil EE OC
Sodium alginate was produced in China ðMv ¼ 7:2 £
(%, w/v) (%, v/v) (%, w/w) (%, w/w)
104 Þ and was purified according to the procedure described
in Section 2.1. The ratio of mannuronic (M) to guluronic (G) E1 2 5 42.0 –
units was determined as 1.27 by 1H NMR spectroscopy E2 4 5 79.9 –
following the procedure described elsewhere (Grasdalen, E3 6 5 87.8 37.5
1983; Grasdalen, Larsen, & Smidsrød, 1979). E4 6 10 92.0 65.0
E5 6 15 88.8 64.2
Pancreatine (Grade VI, 4 £ U.S.P) from swine’s pan-
E6 6 20 89.0 70.1
creas was supplied by Sigma (St Louis, USA) and lipase
 C. Peniche et al. / Food Hydrocolloids 18 (2004) 865–871 867

2.3. Determination of the oil content of the microcapsules appropriate time intervals in order to quantify the oil and
the hexosamines released as the result of the enzymatic
A known weight of microcapsules was grounded in a degradation. Each time the volume was restored with the
Potter homogenizer in the presence of chloroform in order same medium.
to extract the oil content. The mixture was filtered through a
0.45 mm pore sized membrane. The filtrate was placed in a 2.6. Quantification of the oil released
water bath at 37 8C to evaporate the solvent and then placed
in an oven at 40 8C for 1 h in order to ensure complete The total amount of lipids in the aliquots was determined
evaporation of chloroform. The oil was weighed after as described by Pande, Parvin, and Venkitasubra (1963).
cooling. No weight change was detected for periods longer Aliquots taken at different times from the enzymatic
than 1 h. digestion were transferred to test tubes and 2% potassium
dichromate in concentrated sulphuric acid was added (2 ml
2.4. Swelling measurements per ml of sample). The tubes were agitated and heated in a
boiling water bath for 15 min. The tubes were then cooled
Dried capsules were weighed and immersed in excess down and 4.5 ml of water were added to each one. The
distilled water at 25 8C. Water was changed periodically in absorbance of the solution was read at 440 nm. The fatty
order to eliminate low molecular weight electrolytes acids content was evaluated taking as reference a standard
diffusing out of the capsules. The water uptake, W; was curve obtained with stearic acid dissolved in chloroform.
calculated by measuring the weight gain of the sample at (1 mg/ml).
different times after carefully wiping the surface with a filter
paper. It was reported as
2.7. Quantification of hexosamines released
M 2 M0
W¼ ð1Þ
M The hexosamines released during the degradation of the
oil-loaded microcapsules were quantified as described by
where M0 is the weight of the dried capsules and M is that of
the capsules at time t: Elson and Morgan (1993). Aliquots taken at different times
from the enzymatic digestion were transferred to test tubes
2.5. Enzymatic degradation and 6 M HCl was added (2 ml per ml of sample). Hydrolysis
was carried out in boiling water for 4 h. Samples were then
The enzymatic degradation was performed using a neutralized with 3 M NaOH until slightly alkaline and were
modification of the technique presented by Hsu, Vavak, diluted to 10 ml with distilled water. Then 1 ml aliquots of
Satterlee, and Miller (1977) that determines the pH variation each sample were taken and were transferred to test tubes.
resulting from the action of proteolytic enzymes on the One millilitre reagent was added to each tube (1 ml
substrate. The modified technique substitutes the original acetylacetone in 50 ml 1 M Na2CO3) with agitation. The
multi-enzyme system by 6 mg of pancreatine for each tubes were placed in boiling water for 15 min, cooled down
millilitre of water in one determination, and by the direct to room temperature and then their contents transferred to
addition of 1 ml of lipase to the suspension of microcapsules 10 ml flasks. To each flask, 5 ml absolute ethanol and 1 ml
in the other determination. Higher concentrations of enzyme Ehrlich reagent were added and the volume was then
and substrate were also used in the assessment of the release adjusted to 10 ml with absolute ethanol. After 30 min the
of oil and hexosamines during the enzymatic digestion, absorbance of the solutions was read at 530 nm. The
taking into consideration the sensitivity of the method concentration of released hexosamines was determined
employed. The procedure was as follows: two vessels using 0.06 mg/ml of glucosamine chlorhydrate as a
containing 15 ml each of an aqueous suspension of capsules reference. The results were expressed as mg hexosamines/
(6.25 mg/ml) were left to stand until capsules reached mg microcapsules.
hydration equilibrium. The suspensions were adjusted to pH
8 with 0.1 M solutions of HCl or NaOH. Afterwards 5 ml of 2.8. Digestion in Tris – HCl/PBS buffer
enzyme solution (6 mg of pancreatine/ml, at pH 8) was
added to one vessel and 1 ml of lipase to the other. Both A given amount of microcapsules were immersed in
recipients were immediately subjected to magnetic stirring 10 ml of buffer solution. The buffer solution consisted of
at 37 8C and the pH change was monitored during next equal volumes of 1 M Tris –HCl and phosphate buffered
10 min. saline solution (PBS), both at pH 7.4. The buffer containing
Three other vessels were prepared with 15 ml of an the microcapsules was placed in a water bath at 37 8C.
aqueous suspension of capsules (37.5 mg/ml), left to stand Aliquots were taken at different times and the oil released
until the capsules reached hydration equilibrium and were was determined by the method of Pande et al. (1963). In
subjected to the same previously described enzymatic order to mimic the acidic condition in the stomach, in a
procedure. Aliquots were taken from each vessel at second series of experiments the microcapsules were
868 C. Peniche et al. / Food Hydrocolloids 18 (2004) 865–871

pretreated with 0.1 M HCl at pH 1.2 for 4 h, followed by a suspended was 5% of the volume of the sodium alginate
procedure digestion in Tris – HCl/PBS buffer as described. solution.
As shown in Table 1 the EE, expressed as the percentage
2.9. Optical microscopy of shark liver oil entrapped, increased with an increase in
concentration of sodium alginate solution, going from 42%
Surface modifications of capsules were visualized using for the 2% (w/v) sodium alginate solution up to 87.8% for
a Jenalumar microscope (Carl Zeiss, Jena, Germany) the 6% (w/v) solution. It must be pointed out that the
with 63 £ magnification coupled to an Olympus camera. concentration of sodium alginate had also a marked
The diameter of capsules was measured using a stereo- influence on the mechanical stability of the capsules.
microscope (Opton, Carl Zeiss, Jena, Germany) at 50 £ Capsules formed using 2% alginate solution were very
magnification equipped with a graduated ocular lens. weak and loose, whereas capsules prepared with the solution
at 6% (E3 in Table 1) were more compact and had an
appropriate mechanical resistance. In view of these results,
3. Results and discussion the rest of the experiments were performed using 6%
sodium alginate solutions.
3.1. Influence of the chitosan treatment
3.3. Influence of the amount of oil
Sodium alginate capsules were readily permeated by
shark liver oil. As soon as the capsules are isolated the oil Capsules using 6% sodium alginate solutions and
starts to leak due to their porosity. This is illustrated by the different proportions of shark liver oil (experiments E3 to
micrograph in Fig. 1a. The oil exuded from the capsules can E6 in Table 1) were prepared. The EE was as high as 87.8%
be seen as bright yellow. These capsules were obtained or more. However, the content of oil per gram of capsule
under the same experimental conditions as sample E4 in was less than 40% (w/w) in capsules of experiment E3 while
Table 1, but without the chitosan treatment. When capsules it was more than 60% (w/w) in the rest.
are covered with chitosan the oil remains trapped and no It is worth mentioning that capsules E5 and E6 started to
leakage was observed, as shown in Fig. 1b for sample E4. exude the oil 48 h after preparation, while sample E4
This result indicates the importance of coating the retained it adequately, in spite of having similar oil content.
calcium alginate beads with the chitosan/alginate polyelec- It seems that capsules formed from sodium alginate
trolyte complex in order to regulate the permeability of solutions containing more than 10% (v/v) of oil suspended
these capsules. Similar results have been reported for the were not compact enough to retain it adequately.
encapsulation of BSA and insulin in chitosan/alginate
capsules (Hari, Chandy, & Sharma, 1996). 3.4. Swelling of the capsules

3.2. Influence of sodium alginate concentration Because these capsules are used in the swollen state, the
study of their swelling process is of utmost importance in
Capsules were prepared using sodium alginate solutions order to evaluate the suitability of the encapsulation. The
at three different concentrations: 2, 4 and 6% (w/v), dried capsules were swollen in distilled water at 25 8C. In
identified in Table 1 as experiments E1, E2 and E3, Fig. 2 the swelling degree is plotted as a function of time for
respectively. Higher alginate concentrations were not used capsules of experiment E4. There is a rapid increase in the
because their viscosities rendered them very difficult to weight of the capsules up to a maximum value, after which a
handle. In the three cases the volume of shark liver oil slight decrease in the water retention value is observed.

Fig. 1. Optical micrographs (63 £ ) of sodium alginate capsules: (a) without chitosan treatment and (b) after being coated by chitosan. Experimental conditions
are those of experiment E4 in Table 1.
 C. Peniche et al. / Food Hydrocolloids 18 (2004) 865–871 869

Fig. 2. Swelling in water at 25 8C of shark liver oil loaded capsules of Fig. 3. pH variation during the enzymatic digestion of capsules of
experiment E4.
experiment E4 with pancreatine (W) and lipase (X).

A swelling equilibrium value was reached 4 h later. A to the pH decrease was discarded because this parameter
similar pattern in the swelling behaviour has been reported diminished from 8 to 7.5.
for the polyelectrolyte complex between chitosan and In order to obtain more information about the compara-
carboxymethyl cellulose (Argüelles, Hechavarrı́a, Rodrı́- tive enzymatic degradation of these chitosan/alginate
guez, & Peniche, 1993). The equilibrium water content in microcapsules the concentration of glucosamine and fatty
the capsules after 6 h is approximately 45% (w/w). This acids with time was monitored for both enzymes (Fig. 4).
value is very similar the 54% (w/w) reported by Hari et al. In the absence of enzymes, no glucosamine, or fatty acids
(1996) for chitosan/calcium alginate microcapsules. were detected. The lower stability of the microcapsules in
the presence of lipase is evident by the higher increase in the
3.5. Enzymatic degradation concentration of both, fatty acids and hexosamines.

Experiments on the stability and enzymatic degradation

of these microcapsules were further conducted in the
swollen state in order to facilitate the diffusion of ions and
low molecular weight metabolites through the polymer
membrane.
It has been reported that chitosan can be degraded by
proteases. Nishioka et al. (1989) found that chitosan
microspheres release encapsulated material after treating
them with proteases. Furthermore, it has been reported that
chitosan can be hydrolytically degraded by pancreatine and
lipase (Terbojevich, Cosani, & Muzzarelli, 1995). For that
reason, the in vitro stability of microcapsules was tested by
carrying out experiments of enzymatic degradation with
these two enzymes.
The variation of the pH of the medium when a
suspension of microcapsules is submitted to the action of
pancreatine and lipase, respectively, is shown in Fig. 3. In
both cases a decrease in pH is observed, but the effect is
more pronounced with lipase. The increase in acidity is
Fig. 4. Release of hexosamines (circles) and shark liver oil (squares),
attributed to the release of fatty acids present in shark liver expressed as percentage of the estimated total oil in the capsules, during the
oil as the polymer membrane is degraded. It should be enzymatic digestion of capsules with lipase (filled symbols) and
pointed out that a possible effect of polymer solubility due pancreatine (open symbols).
870 C. Peniche et al. / Food Hydrocolloids 18 (2004) 865–871

Table 2 which renders the structure more loose and swellable

Volume changes experienced by chitosan/alginate capsules of experiment modifying the capsule permeability.
E4 loaded with shark liver oil when placed at different pH
The amount of released oil was monitored for 24 h
Treatment Volume (mm3) Swellinga (V/V0) (Fig. 5). The oil was more readily released by the capsules
subjected to a previous treatment at pH 1.2. Moreover, these
Water 3.82 1.8 capsules were readily disintegrated by handling. It is
Buffer (pH ¼ 7.4) 15.30 7.2 important to note that no release of fatty acids was detected
HCl (pH ¼ 1.2) 4.45 2.1
during the four hours of exposure of capsules at pH 1.2.
HCl/Buffer 27.39 12.8
Similar findings were reported by Hari et al. (1996) in a
a
Volume ratio with respect to the dried volume of the capsules similar experiment with chitosan/alginate capsules.
(V0 ¼ 2:14 mm3).

From optical microscopy observations (results not

shown) it is possible to confirm that due to the enzymatic 4. Conclusions
degradation there is a significant deterioration of the walls
of the capsules. This finding corroborates the aforemen- Shark liver oil can be efficiently encapsulated in calcium
tioned results. alginate beads coated with chitosan in order to mask its
unpleasant taste. The chitosan coating allows controlling the
permeability of capsules avoiding oil leakage. These
3.6. Stability of the capsules as a function of pH capsules are degraded by lipase and pancreatine. The
shark liver oil loaded chitosan/calcium alginate capsules are
The conditions of the gastrointestinal tract were initially resistant to the acid environment of the stomach,
modelled by studying the stability of the capsules when but after 4 h at the intestinal pH (7.4), the capsule wall
submitted to different pH environments. One-half of the weakens and they can be easily deteriorated and disinte-
swollen capsules was immersed in Tris –HCl/PBS buffer grated by the mechanical and peristaltic movements of the
(pH ¼ 7.4), while the other half was successively immersed gastrointestinal tract.
in HCl (pH ¼ 1.2) during 4 h and then in the pH 7.4 buffer.
In Table 2, the changes in the swelling degree
experienced by the capsules after those treatments can be Acknowledgements
appreciated. It becomes evident that capsules placed first in
HCl at pH 1.2 followed by buffer experienced a much higher The authors wish to acknowledge the financial support
swelling than in buffer solution alone. This is a consequence from CONACYT, Mexico (‘Programa de Colaboración
of the breakage of polyelectrolyte salt bonds at low pH, México – Cuba’).

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