DESIGNS OF INSULIN-PECTINATE NANOPARTICLES BY ADDITIONAL OF DIFFERENT CROSSLINKER AND COACERVATION AGENT Syed Othman Syed Mohd Al-Azi1,2 , Mokhtar

Mohamad Tarmizi1,2, Sumiran Nurjaya1,2, Tin Wui Wong1,2* 1 Non-Destructive Biomedical and Pharmaceutical Research Centre 2 Particle Design Research Group, Faculty of Pharmacy Universiti Teknologi MARA, Kampus Puncak Alam, Selangor, Malaysia. *wongtinwui@salam.uitm.edu.my Abstract The present study investigated the designs of insulin-pectinate nanoparticles by additional of different crosslinker and coacervation agent. The insulin-pectinate nanoparticles were prepared by the ionotropic gelation method using pectin as the matrix polymer, calcium chloride and zinc chloride as the crosslinker and chitosan as a coacervation agent. The formed nanoparticles were subjected to size and zeta potential, association efficiency and conductivity analysis by using zetasizer, HPLC and conductivity meter respectively. The findings indicated that the insulin-nanoparticles association efficiencies are desirably high with the addition of Ca2+ or Zn2+ crosslinkers but in contrary with coecervation agent, chitosan. The association efficiency values do not correlate with the conductivity of the crosslinkers and coacervation agent. Low association efficiency of insulin-pectinate chitosonium is probably due to its longer chain compared to single Ca2+ or Zn2+ ions. 1. Introduction 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 [3, 7, 8, 9]. Lately, polysaccharides have been utilized to prepare nanoparticulate carrier for delivery of protein drugs through oral route. Nanoparticles, which are defined as colloidal entities 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. Pectinate nanoparticles have been prepared through crosslinking and coacervation the polymer chains with calcium and zinc ions; as well as a chitosan in the liquid state respectively. The present study investigated the effects of different crosslinkers and coacervation agent on the association efficiency of the formed nanoparticles.

2. Materials and methods 2.1 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). 2.2 Formulations of Nanoparticles An aqueous dispersion 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 before introduced dropwise into an aqueous solution containing either 0.05 % (w/w) of calcium chloride dehydrate, 0.01875 % (w/w) of zinc chloride or 0.01 % (w/w) of chitosan in 0.1 % (w/w) acetic.acid The bulk of the solution was subjected to magnetic stirring at 1000 rpm agitation and the stirring was continued for an additional period of 15 min. The formed of insulin-pectinate nanoparticles were subjected to size and zeta potential measurement before separated by stirred ultrafiltration cell (Model 8050, Milipore, USA) fitted with a ultrafiltration membranes (Ultracel PL-100, 100 000 NMWL, 44.5 mm diameter, Milipore, USA). All nanoparticles were dried for 3 days and stored at 4oC subsequently. 3. Results and discussion Table 1 shows the size, zeta potential, AE and conductivity of insulin-pectinate nanoparticles. The freshly prepared nanoparticles had average size, zeta potential and association efficiency of 348 nm, -17.9 mV and 69.8 % respectively for crosslinking with Ca2+. Substitution of Zn2+ did not change the size and zeta potential substantially. The size and zeta potential were increased by 548nm and 82.8mV upon coecervation with chitosan. The association efficiencies are desirably high with the addition of Ca2+ or Zn2+ crosslinkers at 69.8 % and 60.5% but in contrary with chitosan the coecervation agent, which is 1.7%. The association efficiency values do not correlate with the conductivity of the crosslinkers and coacervation agent. Low association efficiency of insulin-pectinate chitosonium is probably due to its longer chain compared to single Ca2+ or Zn2+ ions that hinder its interaction with pectin and insulin. Table 1: Profiles of size, zeta potential and AE for insulin-pectinate nanoparticles and conductivity of crosslinker or coacervation agent. Size (nm) Insulin-pectinate Ca2+ Insulin-pectinate Zn2+ Insulin-pectinate chitosonium 348 12.9
376 76

Zeta potential (mV) -17.90 0.8

-18.5 1.1

AE (%) 69.8 7.1

60.5 15

Conductivity (S/cm) 755.7 0.6

315 2.6

896 90.0

64.9 6.5

1.7 0.0

554 3

4. Conclusion The insulin association efficiency of pectin-insulin nanoparticles can be promoted through croslinking the insulin-pectinate solution with Ca2+ or Zn2+ but not through coacervation with chitosan.

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