MadrasAgric.J.,2024; https://doi.org/10.29321/MAJ.10.500021

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RESEARCH ARTICLE

Received: 13 Aug 2024

Revised: 27 Aug 2024

Accepted: 01 Sep 2024

*Corresponding author's e-mail: drgeethaprincy@gmail.com

Synthesis and characterization of insulin-loaded nanoparticles

fortified milk

P.Geetha^*1 and M.Esther Magdalene Sharon²

Assistant Professor,¹Department of Food Processing Technology,

Assistant Professor,²Dairy Chemistry,

College of Food and Dairy Technology,

Tamil Nadu Veterinary and Animal Sciences University, Chennai-600052,

ABSTRACT

Type 1 diabetes caused by the destruction of the pancreatic cells, leads

to reduced or no production of insulin, the hormone responsible for lowering

blood glucose levels. Insulin is a protein administered subcutaneously to

humans for treating, type I diabetes mellitus to control glucose homeostasis

when pancreatic β-cells production is not sufficient to ensure daily needs of

this hormone. The desire for a more convenient and socially compatible route

of insulin administration other than subcutaneous injection has originated

several approaches to attempt its oral delivery. The aim of this project is

to research and obtain a therapeutic food product containing an oral

insulin delivery system. The method used to synthesize the insulin loaded

nanoparticles is a two step Ionic pre-gelation method for the preparation

of alginate/chitosan nanoparticles. The sizes of the alginate-chitosan

nanoparticles were estimated by Scanning Electron Microcopy to range from

326-850nm. The characterization of the synthesized nanoparticles was

done using Scanning electron microsopy (SEM), Fourier Transform Infrared

spectroscopy (FTIR) and X-Ray Diffraction (XRD) studies. The interaction

between the Alginate and Chitosan was confirmed by the FTIR studies. From

the results of the XRD studies, it was observed that there was a decline in

the crystal structure of the Chitosan after the formation of the nanoparticles.

These Nanoparticles were homogenized with Ultra High Temperature (UHT)

sterilized milk containing 4.5% fat. The standard milk (control) and the milk

containing nanoparticles (product) was subjected to sensory analysis for the

color, aroma and consistency. From the sensory analysis of color, aroma and

consistency, it was found that there was no significant difference between

the control and the product.

Keywords: Alginate, Chitosan, Insulin, Therapeutic Milk, Oral Delivery System

INTRODUCTION

Diabetes is referred to a collection of disorders

defined by elevated blood glucose levels. The condition

arises from an insufficiency in insulin synthesis or

activity, or both, due to various factors, leading to

metabolic abnormalities of proteins and lipids. The

prolonged consequences of hypoglycemia include

harm to tissues and organs. Individuals with type 1

diabetes are incapable of producing sufficient insulin.

This category accounts for around 5%–10% of all

diabetes cases. This type involves the cellular death

of beta cells in the pancreas. In type 1 diabetes, the

pancreas fails to secrete insulin. Insulin is delivered

via subcutaneous injection to manage individuals with

type 1 diabetes (Mobasseri et al., 2020).

In addition to the psychological barriers for the use

of insulin in injectable form, its use is accompanied

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by different complications such as hypoglycemia,

lipoatrophy at the injection site and all other risks

associated with injections. These complications make

the search into alternative routes for insulin delivery

is necessity. The other most studied routes are oral,

nasal, buccal and pulmonary. Buccal route is based

on the micellar solublisation. Nasal route was fully

investigated but no commercial exploitation is taking

place. Pulmonary route was commercially exploited

but unfortunately was withdrawn from the market.

Nevertheless, oral route is the most desired and has

been investigated thoroughly (Elsayed et al., 2011).

Oral delivery system is the preferred route for

administration because it is non-invasive, avoids

injections and decreases the risk of infections. It is also

physiologically desirable since the exogenous protein

imitates the physiological pathway undergoing first

hepatic bypass. The intestinal absorption of proteins

has been reported in a combination of mechanisms

described to explain how the protein cross the intestinal

mucosa (Sarmento et al., 2007). Peptide and protein

drugs are intrinsically poorly absorbable through the

intestinal membrane owing to high molecular weight

and hydrophilicity. Moreover, they are highly susceptible

for enzymatic degradation in the gastrointestinal

(GI) tract after oral administration. Entrapment of

peptides drugs in micro- and nanoparticulate carriers

protects them against the harsh environment of the

GI tract until they are absorbed in released or intact

particular form. In addition, formulations of the carrier

system using mucoadhesive polymers prolongs the

direct contact of the particles to the mucosal surface,

achieves higher local drug concentration in the mucus

layer, and minimizes drug dilution and degradation by

the luminal content (Li et al., 2008).

Nanoparticles

consisting

synthetic

biodegradable polymers, natural biopolymers, lipids

and polysaccharides have been developed and tested

over the past decades. Recently, the idea of using

nanoparticles made from natural biodegradable

polymers to deliver drugs has provoked great

interests. Among them, alginate and chitosan are

very promising and have been widely exploited in

pharmaceutical industry for controlling drug release

(Makhlof et al., 2011). Sodium Alginate has a unique

property of cross linking in the presence of multivalent

cations such as calcium ions in aqueous media.

Alginate forms a reticulated structure in contact with

calcium ions and this network can entrap proteins.

Chitosan is a linear copolymer polysaccharide and it

is produced commercially by deacetylation of chitin,

which is the structural element in the exoskeleton of

the crustaceans. The strong electrostatic interaction of

the amino groups of the chitosan with the carboxylic

groups of the alginate leads to the formation of

the complex chitosan/alginate that becomes the

polyelectrolyte complex between chitosan and alginate

has been widely used in order to obtain microcapsules

for cell encapsulation and devices for the controlled

release of drugs or other substances (Finotelli et al.,

2010).

There is a growing awareness nowadays of

the health benefits of a category of bioactive food

constituents known as nutraceuticals. This has

created society demand for products (functional

foods) and preparations rich in these constituents

to improve public health. It is well documented that

their consumption produces physiological benefits or

reduce the long-term risk of developing degenerative

diseases. On the other hand, the added bioactive

constituents may have some undesirable effects on

the taste and odour of the food matrix used as many of

these constituents have undesirable taste and odour.

Therefore, the objectives of food manufacturers and

nutritionists have been to maximize the availability of

administered nutraceuticals without compromising

consumer acceptability (Salam et al., 2012). This

study aims to develop functional dairy food (milk) with

oral insulin delivery system to overcome the limitations

of the painful subcutaneous injections of insulin

administration and produce a more patient friendly

method of insulin administration. It involves the

synthesis, characterization and mixing of the insulin

loaded nanoparticles into milk and observation of the

settling and dispersion in it.

MATERIAL AND METHODS

Materials

Sodium alginate, chitosan and calcium chloride were

purchased from Himedia Laboratories. Recombinant

human isophane insulin was purchased under the

brand name of Humulin N^®from Biocon Laboratory,

India. Milk used in this study was purchased from the

local market. UHT sterilized standard milk (4.5% fat)

which was marketed by Amul Dairy under the Brand

name of Amul Gold was used in this study. Deioinzed

water (Milli-Q^®) was used throughout the process as a

medium.

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Preparation of Nanoparticles

Insulin loaded Alginate-Chitosan Nanoparticles

were produced by dilute alginate solutions containing

insulin by inducing an ionotropic pre-gel with calcium

counter ions followed by polyelectrolyte complex

coating with Chitosan as described by Sarmento et al,

2007). with isophane insulin as source of insulin.

Nanoparticle Characterization

The prepared nanoparticles were characterized

using scanning electron microscopy, Fourier transform

infrared spectroscopy and x-ray diffraction studies.

The morphology such as the shape, size and the

occurrence of aggregation of particles were studied by

scanning electron microscopy. The interaction between

the various components of the nanoparticulate

systems were studied using Fouriertransform infrared

spectroscopy. The crystalline nature of the components

of the nanoparticles namely, the alginate and the

chitosan were studied using x-ray diffraction studies

before and after the formation of the nanoparticles.

Scanning Electron Microscopy

Scanning electron microscopy was done using a low

vacuum FEI QuantaFEGSEM (5kV) at 80 Torr. Alginate

chitosan nanoparticles were synthesized with three

different concentrations of chitosan. This synthesis

was carried out and the influence of the chitosan

concentration on the size of the nanoparticles formed

was studied using scanning electron microscopy. The

lyophilized nanoparticles powders were mounted on

a stub using a carbon tape and then examined for

the shape, size and the aggregation nature of the

nanoparticles.

Fourier Transform Infrared Spectroscopy

FTIR spectra were measured using a Bruker-α

spectrometer. The samples were gently mixed with

micronized KBr powder and compressed into discs

at a force of 10 kN for 1 minute using a manual

tablet presser. The interferogram was collected in

absorption from 400 cm^-1 and 4000cm^-1 region at

room temperature.

X-Ray Diffraction

X-Ray diffraction of the lyophilized powder samples

was done using PANalyticalXpert Pro XRD in powder

mode. The scanning was done at the rate of 0.05°/

step and 2 sec/step with CuKα as the source of x-ray

with a wavelength of 0.154 nm. The powdered samples

of chitosan, alginate, alginate/chitosan nanoparticles

and insulin loaded alginate/chitosan nanoparticles

were analysed for the crystalline nature.

Association Efficiency (AE)

The association efficiency of the insulin to the

nanoparticles was analysed to find out the amount

of insulin bound to the nanoparticles. The insulin

determinations were done in triplicates. It was

calculated using the following formula:

In-Vitro Insulin Release Studies

The insulin release profile from nanoparticles

in simulated gastric and intestinal pH was carried

out to study the amount of insulin released from

the nanoparticles in to the medium simulating the

stomach and intestinal conditions. The nanoparticles

were placed into test tubes containing 20 mL of HCl

buffer pH 1.5 or PBS buffer pH 7.4. At appropriate

intervals of 30 minutes, aliquots of 500 µL were

taken and replaced by fresh buffer. Aliquots were

centrifuged at 5000g for 15 minutes. The amount of

insulin released from the nanoparticles was evaluated

by HPLC.

Incorporation of nanoparticles into milk

The lyophilized nanoparticles in the form of

powders were added to UHT pasteurized milk. The

total yield of powders from a single batch of prepared

nanoparticles was added to 100 mLmLof UHT

pasteurized milk using a blender. The milk containing

the insulin loaded nanoparticles was kept at 4°C. The

addition of the nanoparticles was done under sterile

conditions to prevent the spoilage of the product.

Sensory analysis

The standard milk and the milk containing insulin

loaded nanoparticles were evaluated for acceptability

by a panel of judges for aroma, color and appearance,

consistency according to 9-point hedonic scale

wherein a score of 1 represented dislike extremely

and score of 9 represented like extremely.

RESULTS AND DISCUSSION

Synthesis and characterization of Insulin loaded

nanoparticles by Spontaneous Emulsification

Solvent Diffusion Method

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Chitosan solutions at concentrations of 0.05%,

0.07%, and 0.09% were prepared for analysis, with

the dimensions and characteristics of the resulting

nanoparticles detailed in Table 1. Each experiment

was conducted in triplicate.

Scanning electron micrographs revealed that

the synthesis process using the lowest chitosan

concentration

(0.05%)

produced

cylindrical

nanofibers, as shown in Figure 1. Table 2 presents the

variance analysis for nanoparticle sizes at chitosan

concentrations of 0.07% and 0.09%, indicating that

the size reduction between these concentrations

was

statistically

insignificant.

The

scanning

electron micrographs of the nanoparticles produced

from the three chitosan concentrations—0.05%,

0.07%, and 0.09%—are shown in Figures 1, 2, and 3,

respectively.

The size of the nanoparticles was measured

to be 68.4±2.800 nm and 66.7±1.201 nm when

synthesizing nanoparticles with starting chitosan

concentrations of 0.07% and 0.09%, respectively.

Particles of a size smaller than 100 nm shown a 2.5-

fold increase in uptake compared to microparticles

measuring 1µm (Panyam and Labhasetwar, 2003).

Preliminary attention was given to polyelectrolyte

interactions and insulin trapping in order to

investigate the linkages between components of the

nanoparticulate systems. The interaction between

the carboxyl (-COO-) group of the anionic polymer

and the amino group (-NH3+) of chitosan is a known

phenomenon. This interaction leads to the formation

of an ionic complex between the two molecules, as

described by Sarmento et al, 2006).

Fourier Transform Infrared (FTIR) spectroscopy

The synthesis of insulin-loaded nanoparticles

was carried out, and the interactions between the

nanoparticle constituents, specifically chitosan and

alginate, were analyzed using Fourier Transform

Infrared (FTIR) spectroscopy. Figure 4 shows the FTIR

spectrum of pure chitosan, which was examined to

identify the highest values within the ranges of 3000–

3120 cm⁻¹, 1480–1530 cm⁻¹, and 1590–1620 cm⁻¹.

These peaks correspond to the presence of amino

groups (-NH₃⁺) in the spectrum. The observed peaks in

Figure 4 confirm the presence of amino groups (-NH₃⁺)

in pure chitosan at wavenumbers of 1501.72 cm⁻¹,

1654.73 cm⁻¹, 3045.94 cm⁻¹, 3079.94 cm⁻¹, and

3147.95 cm⁻¹.

The highest point within the range of 1710 cm^-1

– 1740 cm^-1 indicates the existence of saturated

carboxylic acids. Figure 5 displays a prominent signal

at 1738.31 cm^-1, indicating the existence of the

carboxylic group in the alginate solution employed for

nanoparticle production.

Table 1.The initial concentration of chitosan, size and nature of the nanoparticles

S.No.

Chitosan Concentration%

( w/v)

Size (in nm)

Nature

0.05

Nanofibres like structures

0.07

68.4±2.800

Agglomerated

0.09

66.7±1.201

Agglomerated

Table 2 Peak Values of FTIR spectrum of Alginate, Chitosan, and Nanoparticles formed with initial

chitosan concentrations of 0.05%, 0.07%, and 0.09%.

Samples

Alginate (COO^-) &

Chitosan (NH3

Initial Concentration of Chitosan

Functional groups

0.05%

0.07%

0.09%

COO^--

1738.31

1738.49

1732.83

1742.75

NH3

1501.72

1517.46

1525.96

1514.23

1654.73

1640.73

1606.73

1646.40

3045.94

3284.30

3355.14

2920.16

3079.94

3315.47

3386.31

2952.75

3147.95

3348.05

3416.06

3455.74

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Fig 2. Scanning Electron Micrographs of nanoparticles with initial chitosan concentration of 0.07%

The size of the nanoparticles was measured to be 68.4±2.800 nm and 66.7±1.201 nm when

synthesizing nanoparticles with starting chitosan concentrations of 0.07% and 0.09%, respectively.

Particles of a size smaller than 100 nm shown a 2.5-fold increase in uptake compared to microparticles

measuring 1µm (Panyam and Labhasetwar, 2003).

Fig 3. Scanning Electron Micrographs of Nanoparticles synthesized with initial chitosan concentration of

0.09%

Preliminary attention was given to polyelectrolyte interactions and insulin trapping in order to investigate

the linkages between components of the nanoparticulate systems. The interaction between the carboxyl (-

COO-) group of the anionic polymer and the amino group (-NH3+) of chitosan is a known phenomenon. This

interaction leads to the formation of an ionic complex between the two molecules, as described by

Sarmento et al, 2006).

Fourier Transform Infrared (FTIR) spectroscopy

The synthesis of insulin loaded nanoparticles was carried out and the interactions between the

Fig 3. Scanning Electron Micrographs of Nanoparticles synthesized with initial chitosan

concentration of 0.09%

Volume 111 | Issue 7-9 |

Scanning electron micrographs revealed that the synthesis process using the lowest chitosan

concentration (0.05%) produced cylindrical nanofibers, as shown in Figure 1. Table 2 presents the variance

analysis for nanoparticle sizes at chitosan concentrations of 0.07% and 0.09%, indicating that the size

reduction between these concentrations was statistically insignificant. The scanning electron micrographs

of the nanoparticles produced from the three chitosan concentrations—0.05%, 0.07%, and 0.09%—are

shown in Figures 1, 2, and 3, respectively.

Fig 1.Scanning Electron Micrographs of nanofibre-like structures formed with initial Chitosan Concentration

of 0.05%

Fig 2. Scanning Electron Micrographs of nanoparticles with initial chitosan concentration of 0.07%

Volume 111 | Issue 7-9 |

(

) p

analysis for nanoparticle sizes at chitosan concentrations of 0.07% and 0.09%, indicating that the size

reduction between these concentrations was statistically insignificant. The scanning electron micrographs

of the nanoparticles produced from the three chitosan concentrations—0.05%, 0.07%, and 0.09%—are

shown in Figures 1, 2, and 3, respectively.

Fig 1.Scanning Electron Micrographs of nanofibre-like structures formed with initial Chitosan Concentration

of 0.05%

An analysis was conducted on nanoparticles

synthesized

using

chitosan

concentrations

0.05%, 0.07%, and 0.09% to examine the presence

of amino and carboxyl groups in their respective

FTIR spectra. Figure 6 displays the FTIR spectra of

alginate, chitosan, and nanoparticles prepared with

chitosan concentrations of 0.05%, 0.07%, and 0.09%,

respectively. The peak values for chitosan, alginate,

and the nanoparticles synthesized with initial chitosan

concentrations of 0.05%, 0.07%, and 0.09% are

presented in Table 2. Each spectrum was individually

analyzed to identify any shifts in the peak values of

the amino and carboxyl groups, which would suggest

interactions between chitosan and alginate in the

nanoparticulate systems.

Fig 1.Scanning Electron Micrographs of nanofibre-like structures formed with initial Chitosan

Concentration of 0.05%

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Fig 4. FTIR spectrum of Chitosan

The highest point within the range of 1710 cm^-1 – 1740 cm^-1 indicates the existence of saturated

carboxylic acids. Figure 5 displays a prominent signal at 1738.31 cm^-1, indicating the existence of the

carboxylic group in the alginate solution employed for nanoparticle production.

Figure 5 FTIR spectrum of alginate

An analysis was conducted on nanoparticles synthesized using chitosan concentrations of 0.05%, 0.07%,

and 0.09% to examine the presence of amino and carboxyl groups in their respective FTIR spectra. Figure

6 displays the FTIR spectra of alginate, chitosan, and nanoparticles prepared with chitosan concentrations

of 0.05%, 0.07%, and 0.09%, respectively. The peak values for chitosan, alginate, and the nanoparticles

synthesized with initial chitosan concentrations of 0.05%, 0.07%, and 0.09% are presented in Table 2.

Each spectrum was individually analyzed to identify any shifts in the peak values of the amino and carboxyl

groups, which would suggest interactions between chitosan and alginate in the nanoparticulate systems.

The analysis revealed a slight shift in the peak values of the carboxyl group. Specifically, the peak

shifted from 1738.31 cm⁻¹ in alginate to 1738.49 cm⁻¹ in nanoparticles with an initial chitosan

concentration of 0.05%. Additionally, there was a shift in the peak values of the amino groups, moving from

1501.72 cm⁻¹.

The wavenumber range for chitosan was observed between 1654.73 cm⁻¹ and 3147.95 cm⁻¹,

while for nanoparticles synthesized with a chitosan concentration of 0.05%, the range extended from

1517.46 cm⁻¹ to 3348.05 cm⁻¹.

Volume 111 | Issue 7-9 |

Infrared (FTIR) spectroscopy. Figure 4 shows the FTIR spectrum of pure chitosan, which was examined to

identify the highest values within the ranges of 3000–3120 cm⁻¹, 1480–1530 cm⁻¹, and 1590–1620

cm⁻¹. These peaks correspond to the presence of amino groups (-NH₃⁺) in the spectrum. The observed

peaks in Figure 4 confirm the presence of amino groups (-NH₃⁺) in pure chitosan at wavenumbers of

1501.72 cm⁻¹, 1654.73 cm⁻¹, 3045.94 cm⁻¹, 3079.94 cm⁻¹, and 3147.95 cm⁻¹.

Fig. 5 FTIR spectrum of alginate

The analysis revealed a slight shift in the peak

values of the carboxyl group. Specifically, the peak

shifted from 1738.31 cm⁻¹ in alginate to 1738.49 cm⁻¹

in nanoparticles with an initial chitosan concentration

of 0.05%. Additionally, there was a shift in the peak

values of the amino groups, moving from 1501.72

cm⁻¹.

The wavenumber range for chitosan was observed

between 1654.73 cm⁻¹ and 3147.95 cm⁻¹, while

for nanoparticles synthesized with a chitosan

concentration of 0.05%, the range extended from

1517.46 cm⁻¹ to 3348.05 cm⁻¹.

The spectra indicate a shift in the peak values

of the carboxyl group, moving from 1738.31 cm⁻¹ in

alginate to 1732.38 cm⁻¹ in nanoparticles with an

initial chitosan concentration of 0.05%. Similarly,

shifts are observed in the peak values of the amino

groups, which change from 1501.72 cm⁻¹, 1654.73

cm⁻¹, 3045.94 cm⁻¹, 3079.94 cm⁻¹, and 3147.95

cm⁻¹ in chitosan to 1525.96 cm⁻¹, 1606.73 cm⁻¹,

3355.14 cm⁻¹, 3386.47 cm⁻¹, and 3416.06 cm⁻¹ in

nanoparticles formed with a chitosan concentration of

0.07%.

For nanoparticles prepared with an initial chitosan

concentration of 0.09%, the carboxyl group peak

shifted from 1738.31 cm⁻¹ in alginate to 1742.75

cm⁻¹ in the nanoparticles. The amino group peaks

also displayed changes, moving from 1501.72 cm⁻¹,

1654.73 cm⁻¹, 3045.94 cm⁻¹, 3079.94 cm⁻¹, and

3147.95 cm⁻¹ in chitosan to 1514.23 cm⁻¹, 1646.40

cm⁻¹, 2920.16 cm⁻¹, 2952.75 cm⁻¹, and 3455.74

cm⁻¹ in the nanoparticles, respectively.

The results of the study indicate that the shifts in the

peaks representing the amino group and the carboxyl

groups in the FTIR spectrum of the nanoparticles

provide evidence of the interaction between the

carboxyl (-COO-) group of the anionic polymer and the

amino group (-NH3+) of Chitosan. This interaction

leads to the formation of an ionic complex between

the two compounds in the nanoparticulate system.

X-ray diffraction (XRD) studies

The nanoparticles containing Humulin N, along

with

the

nanoparticulate

system

components,

chitosan and alginate, were analyzed using X-ray

diffraction (XRD) to assess any

Fig 4. FTIR spectrum of Chitosan