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Mohd 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. *


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)

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. 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

Fig. 1 Schematic representation of a) pectin-chitosan b) pectin-crosslinker (Ca2+ or Zn2+) interaction. [1]

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. Crosslinkers Size (nm) 348.0 12.9 Calcium 376.0 76.0 Zinc Chitosan Zinc 300x 896.0 90.0 64.9 6.5 Calcium 1.7 0.0 554.0 3.0 Chitosan -18.5 1.1 Zeta potential (mV) -17.9 0.8 69.8 7.1 60.5 9.5 755.7 0.6 315.0 2.6 Association efficiency (%) Conductivity (S/cm)

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).
Crosslinking or coacervating agent Extrusio n Pectin + insulin Dispersion transferred to petri dish



Fig. 3. SEM profiles for insulin-pectinate nanoparticulate films.

Conductivity measurement

The difference in insulin association efficiency of nanoparticles was not ascribed to the
Drying of dispersion into film at 4C Filtration

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

Redispersi on

Scanning Electron Microscopy

underwent a lower degree of densification. (Table 1; Fig. 3).

REFERENCES [1] Silpakorn University International Journal, Vol. 3 pp. 206-228,

Size and zeta potential measurement

Association Efficiency by High Performance Liquid Chromatography

(Number 1-2) 2003. [2] Carbohydrate Polymers., vol. 62, pp. 245-257, 2005. [3] Eur. J. Pharm. Biopharm., vol. 69, pp. 176188, 2008. [4] Drug Dev. Ind. Pharm., 30,359-367.

Fig. 2: Workflow of nanoparticles and nanoparticulate films preparation