Characteristics and Release Profile:

Formula of Insulin Nanoparticles using Medium Molecular Weight Chitosan and Pectin Polymers


Tiara Mega Kusuma1*, Teuku Nanda Saifullah Sulaiman2, Ronny Martien2

1Department of Pharmaceutics, Faculty of Health Science, Universitas Muhammadiyah Magelang, Indonesia.

2Department of Pharmaceutics, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia

*Corresponding Author E-mail:



Insulin is a macromolecular polypeptide hormone with low drug stability and permeability along the digestive tract. The nanoparticle delivery system has been proven to be able to increase the bioavailability of per-oral insulin. However, the formulation of insulin nanoparticles using chitosan and pectin polymers has not been widely studied. The purpose of this research is to figure out the physical characteristics and profile of insulin release from nanoparticle formulas made with ionic gelation techniques using chitosan and pectin polymers. The 0.1% insulin nanoparticle formula is made with variations of 2 levels of medium molecular chitosan and pectin concentrations to obtain 4 formulas, i.e. F1 (0.01%; 0.1%), F2 (0.03%; 0.1%), F3 (0.01%; 0.2%), and F4 (0.03%; 0.2%). The optimum formula is determined by the factorial design method contained in the Design Expert program using response characteristics in the form of percentage of the entrapment efficiency and zeta potential value. The selected formula is then tested for particle size and shape, and insulin release profile in vitro. The particle size and morphology are observed with TEM (Transmission Electron Microscope), while the insulin release profile is determined on HCl buffer media pH 1.2 and PBS pH 6.8. The optimization results of the formula show that F1 is the optimum formula with a desirability value of 0.786. The selected formula shows that the entrapment efficiency is 57.66%, the zeta potential is 12.0 mV, the shape of particles is spherical, and the size is <500 nm. In vitro studies show the profile of insulin release from the matrix following the Weibull kinetics model on HCl and Korsmeyer-Peppas media on PBS media, using the Fickian diffusion method. Overall, the insulin nanoparticles obtained have met the expected characteristic of the nanoparticles.


KEYWORDS: Insulin, Chitosan, Nanoparticle, Ionic gelation.




Per-oral drugs have the easiest route, and they are the most comfortable drugs to be used by patients. Nevertheless, not all drugs are able to be given orally dealing with the physicochemical characteristics of the drug, as well as its ability to counter the physical and chemical barrier along the digesting system1 that decreases the bioavailability of the drug. Many drugs have low solubility and/or permeability level1,2, one of which is insulin.


This fact challenges researchers to be able to develop insulin become per-oral drug with high bioavailability according to the expected effect of the therapy3.


Insulin is polypeptide hormone used by injection as the main therapy of DM (Diabetes Mellitus) type 1 and 2, which the blood glucose level cannot be controlled by oral anti-diabetes drugs4,5. Insulin is a macromolecule with a size of 5700 Dalton, so the solubility and permeability of the drug is low in the digestive tract. Besides, it is also easy to be degraded by various enzymes in the digestive tract, such as protease that causes only a small amount of the drug absorbed6. Based on BCS (Biopharmaceutical Classification System) approach, insulin is suitable to be developed to become oral medication in form of nanoparticle7. Nanoparticle of oral insulin is known can enhance bioavailability of per-oral insulin through the decrease of permeability and stability of insulin in the digestive tract6,8,9.


The making of nanoparticles with ionic gelation technique is widely applied on macromolecular drugs as it is proven to be able to absorb active substance with wider characteristics. The formulation of nanoparticle with chitosan as the polycation is the most frequently used polymer due to its characters, which is not toxic, and it has potential in delivering drug, protein, peptide, and DNA10. Chitosan has characteristics that other polymers do not have. They are the ability to enhance drug’s transportation in paracellular chamber through tight junction cell opening, and the ability to increase drug’s stability from the damage of enzyme and pH in the digestive tract11–13, increasing mucoadhesive, as well as refining and lengthening the drug absorption in the intestine14. Chitosan with medium molecular weight has stronger electrostatic bond with DNA, which make the drug loading and bigger drug absorption possible, and it can lengthen and decrease the release of insulin from the matrix better than chitosan with low molecular         weight15–17.


A negatively charged polymer that is frequently used is pectin since its nature that can control the release of drug well in the colon18,19. Complex polyelectrolyte from chitosan-pectin-gum Arabic has an ability to act as stable and well controlled drug delivery agent20,21. Interaction between polycationic chitosan and polyanionic pectin with ionic gelation technique will result in  100-400nm particles, which are relatively stable with zeta potential value > 30mV, which is good for protein drug delivery system16,22. The making of nanoparticles with combination of chitosan with medium molecular weight polymer and pectin is also known can result bigger zeta potential with higher stability of colloidal nanoparticle dispersion system.


The nanoparticle formulation of insulin uses matrix polymer using chitosan-insulin10, chitosan-gum-         arabic10,21, chitosan-alginate9, chitosan-TPP (tripolyphosphate)23 has been studied widely, but the making of insulin nanoparticle with chitosan and pectin has not been done a lot. The combination of medium molecular weight polymer chitosan and pectin has huge potential as good matrix to produce nanoparticle of insulin with ionic gelation technique. Development of insulin in form of non-invasive preparations, such as per-oral, is necessary to actualize routine therapy4.



Chemicals and reagents:

Insulin bovine pancreas (C254H377N65O75S6) purity ≥ 27 USP unit/mg (Beijing TOP Science Biotechnology Co., Ltd), medium molecular weight chitosan (C6H11NO4)n beige powder from crab skin with viscosity 503 cps and deacetylation degree 80% (Sigma-Aldrich), pectin from apple (Sigma-Aldrich®), Reagen Bio-rad Protein Assay (Bio-Rad®), HCl 37%, glacial acetic acid (Merck), demineral water (Brataco).



Sspectrophotometer UV-Vis (Genesys 10S, US), Transmission Electron Microscope (TEM) (JOEL-JEM 1400, Japan), Particle Size and Zeta Potential Analyzer (SZ- 100 Horiba Scientific), Shaking Thermostatic Waterbath (Julabo, U3), pH meter (Hanna), magnetic stirrer, hot plate stirrer and vortex mixer (MaxiMixTM), analytical scale (Mettler Toledo), micropipette 100μL–1000μL (Biologix, USA), microtube eppendorf 1,5mL; conical tube 15mL (Labtip, India), refrigerated centrifuge (Velocity 18R).


Preparation of nanoparticles:

All of the materials are mixed in a form of solution, in which insulin, chitosan, and pectin are dissolved in 0.01N HCL; 1% acetic acid; and aquademineral. Insulin solution is made in pH 4.0; while chitosan and pectin in pH 5.0.  The formula of insulin nanoparticle is made with mixing technique that uses high energy with vortex mixer using volume ratio of chitosan, insulin, and pectin (1:1:1). The design of insulin nanoparticle formula is presented (Table 1).


Table 1: Formula of insulin nanoparticles






0,01 %

0,1 %

0,1 %


0,03 %

0,1 %

0,1 %


0,01 %

0,1 %

0,2 %


0,03 %

0,1 %

0,2 %


Analysis of entrapment efficiency

The insulin concentrate is analyzed using Bradford method. The sample is centrifuged in 15,000rpm of speed with temperature of 4°C for 50 minutes. 50μL supernatant is dissolved with750μL aquademineral. Add 200μL of biorad and incubate for 10 minutes in room temperature. The measurement of absorbance is with spectrophotometry UV-Vis at λ 595nm.


                                                Total insulin-free insulin

Entrapment efficiency (%) = –––––––––––––––––––x100%

                                                            Total insulin


Zeta potential determination:

The zeta potential of insulin nanoparticle is determined using Zeta Potential Analyzer with Electrophoretic Light Scattering method.


Optimization of insulin nanoparticle formula:

The optimization of the formula uses Factorial Design of Design Expert®9 software. The measured responses are the data of the measurement of the entrapment efficiency and zeta potential. The optimum formula is determined according to the highest desirability value.

Morphology of particles:

The size and shape of the particles is inspected using test tool TEM (Transmission Electron Microscope).


In vitro release profile:

Insulin release from a preparation is tested In vitro by using shaking thermostatic water-bath tool (Jubalo, U3) and using simulation media of intestine (Phosphate-buffered saline pH 6.8) and stomach (Buffer HCl pH 1.2) without enzyme effect with shaking speed 50rpm in temperature of 37°C for 4 hours. In the 15th, 30th, 45th, 60th, 90th, 120th, 180th, and 240th minutes, the samples are taken to later be centrifuged and the insulin level in the supernatant is analyzed.



Nanoparticles characterisation respon:

Entrapment efficiency obtained from the four formulas is in the range of 49.19 – 57.66 (%) with the insulin in nanoparticle as much as 470 – 590 (μg) in 1.5ml of sample. One IU insulin is as much as 45.5μg crystal insulin, so the insulin content in 1.5ml nanoparticle sample is as much as 10.32 – 12.97 IU (Table 2).


Table 2: Characterization of Nanoparticle of Insulin

Characteristic respon






Entrapment efficiency (%)





Zeta potential (mV)






Formula optimum:

The prediction using Design Expert®9 program obtains optimum formula of nanoparticle insulin in F1 using medium molecular weight chitosan and pectin with low concentration in which the predicted characteristic of the nanoparticle obtained is 72.6% suitable with the characteristics expected. The One Sample T-Test analysis obtains significance value higher than 0.05, which means there are no significant differences between the result obtained and the result expected (Fig.1).



Fig.1: Desirability value of Nanoparticle


Morphology of particles:

The result of a test using TEM shows that the particle from nanoparticle system insulin produced is spherical (Fig. 2), slightly white misty with size less than 500nm


Fig. 2: Nanoparticle morphology


Insulin Release Profiles:

Insulin release on buffer media HCl pH 1.2 follows the kinetic model of Weibull with diffusion process that follows the law of Ficks. While on buffer media of phosphate saline pH 6.8, it follows Korsmeyer-Peppas with Fickian diffusion mechanism (Fig.3).


Table 3: Insulin Release





HCl 1.2




PBS 6.8




AIC   : Akaike Information Criterion

MSC  : Model Selection Criterion


Fig.3: Insulin release profiles



Insulin is a macromolecular hormone from protein group that contains 51 amino acid ordered in two chains (A and B) and connected by disulfide bond24. Insulin has firm stability in the refrigerator and is protected from freezing that can change the structure of the protein and reduce the potential for insulin25. Insulin activity can be lost due to several factors such as esterification of carboxyl groups, oxidation or reduction, destruction by proteolytic enzymes such as kemotripsin and pepsin, especially enzymes that can degrade insulin, namely glutathione insulin transhydrogenase which uses reduced glutathione to break down disulfide bridges, modification of free amino groups, especially enzymes that can degrade insulin, namely glutathione insulin transhydrogenase which uses reduced glutathione to break down disulfide bridges, modification of free amino groups or aliphatic hydroxyl groups26. The isoelectric point of insulin is at pH 5.3. The isoelectric point is a degree of acidity or pH value where the macromolecules (proteins) have zero charge and have the same number of negative charges as the number of positive charges. An amino acid is characterized by the presence of an amino group (- NH3 +) which is basic and a carboxyl group (-COO) that is acidic. The presence of two plus and minus ions in amino acids makes insulin dipolar (Zwitter Ion). In colloids, if the pH is made equal to the isoelectric point, then some or all of the charge on the particles will be lost during the ionization process (neutral charge). If the pH is below the isoelectric point, the colloidal particle charge will be positively charged, and vice versa.


The ability of nanoparticles to increase the bioavailability of protein-based drugs in the systemic has been proven by various mechanisms, such as increased solubility, permeability, and stability along the digestive tract27–29. Nanoparticles made by ionic gelation techniques using cationic and anionic polymers are preferred because they are relatively easy, inexpensive, and fast in their manufacturing process, but are capable of producing good nanoparticle characteristics30. The principle of making nanoparticles with ionic gelation techniques is to use polysaccharide polymers that have opposite charges to form ionic bonds so that they can form a flexible matrix through electrostatic bonds to absorb drugs with wider properties, for example a combination of chitosan-alginate; chitosan- gum-arabic, chitosan-TPP19,31–35.


The making of nanoparticles in this research uses chitosan as the positively charged polymer and negatively charged pectin. The complexations of chitosan and pectin show high mucoadhesive character with good drug release in the colon19,36. Chitosan is positively charged polysaccharide with zeta potential value 94,8mV in acid pH solution, while pectin is negatively charge polysaccharide19 with zeta potential value -22.0mV when dissolved in acidic pH solution21. Chitosan in solution with acidic pH change the amine group structure (-NH2) to be positively ionized (-NH2). Positive ionized groups can form ionic interaction with pectin that contains carboxyl group (COO-) 33,37,38. Ionic interaction between chitosan and pectin can form polymer matrix with particles’ size in nanometer-scale dispersed in solution. Visually, the forming of nanoparticle is indicated by the presence of opaque, a relatively transparent colloid dispersion. It is as the result of mass reduction of each particle so that the width of the surface is gain and resulting in repulsive interaction among bigger particles and the presence of the phenomenon of Brown move as one of the specific characters of particles in colloidal size20.


The result of entrapment efficiency test of insulin in polymer base shows that chitosan and pectin concentration increase causes significantly decrease of the percentage of entrapment efficiency with p-value 0.0005 and 0.0454. Entrapment efficiency test is used to figure out how big the percentage of active substances of insulin that can be absorbed in chitosan and pectin matrix is. The previous research showed that the increase of chitosan concentration could decrease the percentage of the efficiency because chitosan allegedly could obstruct the ionic interaction between insulin and          pectin9,14,31. The greater the concentration of chitosan, the greater the amount of pectin that binds chitosan than that binds with insulin. As a result, the release of insulin is greater and the efficiency of absorption is smaller. In addition, because insulin in this experiment is made at pH 4.0 (below the isoelectric point) so it will tend to be positively charged so that when it is mixed with chitosan, which is also positively charged, the ionic interactions that occur are not too strong, so the amount of insulin absorbed in chitosan is also small.


Meanwhile, the zeta potential test reveals that chitosan has positive response to zeta potential, while pectin significantly has negative response to zeta potential with p-values respectively 0.0044 and 0.0001. The increase of the concentration of chitosan will raise the zeta potential value because of the widening distance between the stern layer and the diffuse layer so that the potential difference is greater and the attractive force between opposing charged particles is reduced which can increase the stability of the dispersion system. The increase of the concentration of pectin can reduce the zeta potential, presumably because an increase in the excess negative charge in the system provides an attractive force with a positive charge from the particle, thereby reducing the zeta potential value. The formula of insulin nanoparticles obtained has a positive charge. It is assumed that the surface of the particle is dominated by positive charge originating from chitosan. Positive surface charge particles in the nanoparticle system increase insulin uptake in the mechanism of opening the tight junction cells that are negatively charged.


The entrapment efficiency and zeta potential test results used as response characteristics in the optimization of insulin nanoparticles formula obtain the optimum formula of insulin nanoparticles in F1 with the use of chitosan and low concentrations of pectin with a desirability predictive value of 0.786. Optimization of the insulin nanoparticle formula is needed to determine the exact composition of chitosan and pectin concentrations based on the optimum conditions of response characteristics, the interaction of factors on the response, and the greatest effect of the factors affecting the response. Contour plots of the test parameters are combined to obtain a graph showing the optimum formula. The color in the contour plot shows the prediction of the influence of factors on the desirability value. The red color indicates the greater desirability value.


The result of the test using TEM (Transmission Electron Microscope) on the optimum formula shows spherical and a little bit misty white particles with size less than 500nm. It is allegedly due to the lack of the cross-link power between positively charged chitosan and negatively charged pectin through the electrostatic bond. The increase in pectin concentration is thought to reduce particle size because it provides stronger cross links with chitosan so that the resulting particles are more compact and smaller in size. Chitosan has good absorbent nature with the opposite particle load, in this case is pectin. The presence of white mist in the chitosan matrix is assumed to be free insulin in the nanoparticle system or insulin that attaches to the surface of the polymer matrix. The spherical shaped particles show that chitosan absorbs insulin and forms a cross link with pectin through its electrostatic bonds quite well39. While the particle size obtained from the selected formula is categorized quite large, but this size is still included in the category of nanoparticles where the particle size is in the range of 1-1000nm40. However, this limitation is difficult to reach for nanoparticle system as the drug delivery system as, generally, there supposed to be sufficient content of drug in matrix in each particle, so that relatively bigger size is required28. Size and shape of particles are the most important characteristics in nanoparticle system as they affect drug loading, drug release, and the stability of nanoparticle system41.


Polymers of chitosan and pectin have important roles in improving drug stability in digestive tract so that the amount of drug released can be minimized. The ability of the matrix system of chitosan and pectin polymer in protecting insulin is tested in vitro by making dissolution media that suitable with the condition of stomach and intestines19,20,42. In stomach it is simulated by making media of HCl buffer pH 1.2, while in the intestines it is simulated using media of PBS buffer pH 6.8. The test is done by ignoring the effect of enzymes. The determination of the profile of insulin release in a media is by seeing the value of R2adjusted that closest to 1, the smallest AIC (Akaike Information Criterion) value, and the biggest MSC (Model Selection Criterion) value. Negative MSC value indicates that the data have range that is far from the model. The release of insulin from the polymer matrix system in the acid medium follows the Weibull model, whereas in the base medium it follows the Korsmeyer-Peppas model with the Fickian diffusion mechanism. The diffusion process with Ficks Law shows the release of insulin that occurs through a swelling mechanism where the dissolution medium enters the polymer matrix system, then dissolves insulin in the matrix and transports dissolved insulin out of the matrix through the pores. The process of diffusion of the medium into the polymer matrix system is also assumed to depend on the degree of crosslinking strength between chitosan and pectin as the system.



The insulin nanoparticle formula produced using medium molecular weight chitosan polymers and pectin with ionic gelation techniques has the potential to be developed as a promising oral preparation to achieve high bioavailability because it produces characteristics and the release of insulin is quite good. However, this study still has limitations in which insulin release tests are carried out on media without the influence of enzymes. Even though one of the factors limiting the amount of drug absorbed is due to the low stability of the enzyme effect.



The authors are grateful to the authorities of Faculty of Pharmacy, Universitas Gadjah Mada for the facilities.



The authors declare no conflict of interest.



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Received on 14.01.2020            Modified on 16.05.2020

Accepted on 03.08.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):1973-1978.

DOI: 10.52711/0974-360X.2021.00349