INSULIN-PECTINATE NANOPARTICLES PREPARED BY IONOTROPIC GELATION AND COACERVATION METHODS
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INSULIN-PECTINATE NANOPARTICLES PREPARED BY IONOTROPIC GELATION AND
COACERVATION METHODSMohd Mokhtar Mohammad Tarmizi, Syed Othman Syed Al-Azi, Sumiran Nurjaya, Tin Wui Wong*
Particle Design Research Group,
Non-Destructive Biomedical and Pharmaceutical Research Centre, Faculty of Pharmacy,
Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia.
Fig. 1 Schematic representation of a) pectin-chitosan b) pectin-crosslinker (Ca2+ or Zn2+) interaction. [1]
MATERIALS AND METHOD
Materials
Pectin (methoxy content=9.0%, galacturonic acid content=87.6%, Sigma Aldrich, USA)
was employed as matrix polymer in the preparation of nanoparticles, with calcium
chloride dihydrate (Merck, Germany) and zinc chloride (Merck, Germany) as crosslinker
as well as chitosan (degree of deacetylation=86%, Zulat Pharmacy, Malaysia) as a
coacervation agent. Other chemicals included hydrochloric acid, acetic acid and sodium
hydroxide (Merck, Germany).
Methods
Preparation of Nanoparticles
An aqueous solution containing 0.1 % (w/w) of pectin and 0.015 % (w/w) of insulin in 0.01
M HCl was adjusted to pH 3.0 by using 0.5 M NaOH and introduced dropwise into an
aqueous solution containing either 0.05 % (w/w) of calcium chloride dihydrate, 0.01875 %
(w/w) of zinc chloride or 0.01 % (w/w) of chitosan in 0.1 % (v/v) acetic acid (Fig. 2). The
bulk of the dispersion was subjected to magnetic stirring at 1000 rpm agitation. The
formed insulin-pectinate nanoparticles were subjected to size and zeta potential analysis
using dynamic light scattering and electrophoretic light scattering techniques respectively
at 25C in triplicates (Malvern, UK). The insulin association efficiency, defined as the
quotient of amount of encapsulated insulin to theoretical amount of insulin employed in
the preparation of nanoparticles, in the unit of percentage was analyzed by means of high
performance liquid chromatography (Agilent, USA).
RESULTS AND DISCUSSIONS
The negatively charged Ca2+ and Zn2+ crosslinked pectinate nanoparticles had smaller
sizes of 348.0 12.9 and 376.0 76.0 nm respectively than the positively charged
chitosan coacervated pectinate nanoparticles (896.0 90.0 nm). The pectinate
nanoparticles demonstrated a high insulin association efficiency when Ca2+ and Zn2+
were used as a crosslinking agents (> 60%), whereas chitosan-pectinate coacervate
had a low insulin association efficiency (< 2%) (Table 1). The insulin association
efficiency of pectinate nanoparticles was not directly correlated with the conductivity of
crosslinking and coacervation agents, which presumably could govern the rate of
nanoparticle formation and insulin encapsulation (Pearson correlation, r=0.077,
p>0.05).Table 1: Profiles of size, zeta potential and association efficiency for insulin-pectinate nanoparticles
and conductivity of crosslinker or coacervation agent.
Fig. 3. SEM profiles for insulin-pectinate nanoparticulate films.
CONCLUSION
The difference in insulin association efficiency of nanoparticles was not ascribed to the
variation of conductivity of crosslinking or coacervating agent. Low insulin association
efficiency of chitosan coacervated pectinate nanoparticles was attributed to porous
nature of coacervate which permitted insulin loss during the process of
nanoparticulation. This was inferred from the larger physical size of coacervate which
underwent a lower degree of densification. (Table 1; Fig. 3).
Zinc Calcium Chitosan
300x
1000x
2000x
Pectin +insulin
Association Efficiency by HighPerformance
Liquid Chromatography
Scanning ElectronMicroscopy
Drying of dispersioninto film at 4C
Size and zeta potentialmeasurement
Filtration
Conductivitymeasurement
Extrusion
Dispersion transferredto petri dish
Crosslinking orcoacervatingagent
Redispersion
In Association With
PARTICLE DESIGN RESEARCH
GROUP
UNIVERSITI TEKNOLOGI
MARA
NON-DESTRUCTIVE
BIOMEDICAL
AND PHARMACEUTICAL
RESEARCH CENTRE
INTRODUCTION
Nanoparticles are nano-sized particles that are made up of a shell and a space specifically formulated to carry drugs. The formation of nanoparticles can be achieved by several
techniques namely ionotropic gelation, coacervation and others. Frequently, nanoparticles are fabricated using polysaccharides. Natural polysaccharides, such as pectin and
alginate, are widely employed in the preparation of pharmaceutical solid dosage forms due to their non-toxic, biodegradable, biocompatible and hydrophilic characteristics [1-3].
Nanoparticles, with sizes ranging between 10-1000 nm, can protect the protein drugs against enzymatic and hydrolytic degradation as well as control their release patterns in the
gastrointestinal tract. Insulin as a type of protein drug is susceptible to degradation by proteolysis activity of the gastrointestinal tract [4]. In the present study, insulin-pectinate
nanoparticles have been prepared by ionotropic gelation and coacervation process using calcium chloride and zinc chloride as crosslinking agents and chitosan as coacervation
agent (Fig. 1). The formed nanoparticles were subjected to size, zeta potential, insulin association efficiency and scanning electron microscopy analysis. The reactivity of
crosslinking and coacervation agents in liquid phase was illustrated by their conductivity values.
a) b)
Crosslinkers Size (nm) Zeta potential(mV)
Associationefficiency (%)
Conductivity(S/cm)
Calcium348.0 12.9 -17.9 0.8
69.8 7.1 755.7 0.6
Zinc376.0 76.0 -18.5 1.1 60.5 9.5 315.0 2.6
Chitosan 896.0 90.0 64.9 6.5 1.7 0.0 554.0 3.0
REFERENCES
[1] Silpakorn University International Journal, Vol. 3 pp. 206-228,
(Number 1-2) 2003.
[2] Carbohydrate Polymers., vol. 62, pp. 245-257, 2005.
[3] Eur. J. Pharm. Biopharm., vol. 69, pp. 176188, 2008.
4 Dru Dev.Ind. Pharm. 30359-367.
Fig. 2: Workflow of nanoparticles and nanoparticulate films preparation
Preparation of film from nanoparticulate dispersion
The same dispersion was subjected to drying at 4C in petri dish. The formed film was
collected and subjected to Field Emission Scanning Electron Microscopy (SEM, Jeol,
Japan) analysis.
