Concanavalin A conjugated biodegradable nanoparticles for oral insulin delivery

RESEARCH PAPER

Concanavalin A conjugated biodegradable nanoparticlesfor oral insulin delivery

Pooja Hurkat • Aviral Jain • Ashish Jain •

Satish Shilpi • Arvind Gulbake • Sanjay K. Jain

Received: 27 May 2012 / Accepted: 24 September 2012 / Published online: 10 October 2012

� Springer Science+Business Media Dordrecht 2012

Abstract Major research issues in oral protein

delivery include the stabilization of protein in delivery

devices which could increase its oral bioavailability.

The study deals with development of oral insulin

delivery system utilizing biodegradable poly(lactic-

co-glycolic acid) (PLGA) nanoparticles and modify-

ing its surface with Concanavalin A to increase

lymphatic uptake. Surface-modified PLGA nanopar-

ticles were characterized for conjugation efficiency of

ligand, shape and surface morphology, particle size,

zeta potential, polydispersity index, entrapment effi-

ciency, and in vitro drug release. Stability of insulin in

the developed formulation was confirmed by SDS-

PAGE, and integrity of entrapped insulin was assessed

using circular dichroism spectrum. Ex vivo study was

performed on Wistar rats, which exhibited the higher

intestinal uptake of Con A conjugated nanoparticles.

In vivo study performed on streptozotocin-induced

diabetic rats which indicate that a surface-modified

nanoparticle reduces blood glucose level effectively

within 4 h of its oral administration. In conclusion, the

present work resulted in successful production of Con

A NPs bearing insulin with sustained release profile,

and better absorption and stability. The Con A NPs

showed high insulin uptake, due to its relative high

affinity for non-reducing carbohydrate residues i.e.,

fucose present on M cells and have the potential for

oral insulin delivery in effective management of Type

1 diabetes condition.

Keywords Concanavalin A � Biodegradable

nanoparticles � Protein � PLGA � Insulin �Oral delivery

Introduction

Diabetes mellitus was known in antiquity and remains

today a worldwide and increasing health problem. It

occurs because of lack of insulin, with or without

factors that oppose the action of insulin. The beta cells

of pancreas are decreased in number or are degranu-

lated in diabetes. The reduction in number of beta cells

corresponds to the lack of insulin. In Type1 diabetes

there are no beta cells, in Type 2 diabetes only about

one half of them are present. Although insulin

deficiency is the primary defect in IDDM (Insulin

dependent diabetes mellitus), in patients with poorly

controlled IDDM there is also a defect in the ability

of target tissues to respond to the administration of

insulin.

IDDM patients require insulin to keep their blood

sugar levels under tight control. The need for regular

daily injections is a major drawback for diabetic

P. Hurkat � A. Jain � A. Jain � S. Shilpi �A. Gulbake � S. K. Jain (&)

Pharmaceutics Research Projects Laboratory, Department

of Pharmaceutical Sciences, Dr. Hari Singh Gour

Vishwavidyalaya, Sagar 470003, MP, India

e-mail: [email protected]

123

J Nanopart Res (2012) 14:1219

DOI 10.1007/s11051-012-1219-4

patients and can reduce compliance with treatment.

Considerable research efforts have been devoted to the

development of alternative modes of insulin delivery

that are safe, effective, and do not involve injection

(Owens 2002). Various alternatives approaches to

injectable insulin, which present advantages of avoid-

ing pain and discomfort, have been investigated

including: insulin patches (Shaikh et al. 2005; Cevc

1996), insulin pumps, and inhaled insulin (Chen et al.

2002; Desai et al. 2002; Johannson et al. 2002;

Pringels et al. 2006). Some noninvasive routes like

buccal delivery (Veuillez et al. 2001, Xu et al. 2002,

Portero et al. 2007, Das et al. 2010), vaginal delivery

(Ning et al. 2005), oral delivery (Chalasani et al. 2007,

Socha et al. 2009, Sonaje et al. 2010, Wua et al. 2012),

ocular delivery (Srinivasan and Jain 1998, Xuan et al.

2005, Diebolda et al. 2007, Motwani et al. 2008, Zhu

et al. 2012), etc. also have been investigated.

Designing oral insulin delivery in therapy of Type 1

patients has been a persistent challenge due to several

unfavorable physicochemical properties of peptide

and proteins including large molecular size, suscep-

tibility to enzymatic degradation, short plasma half-

life, ion permeability, immunogenicity, and tendency

to aggregate, adsorption, and denaturation (Mahato

et al. 2003). Moreover, there are various physiological

variables that influence, and more often that hinder

the oral bioavailability of protein and peptide drugs,

such as gastric emptying, gastric transit, the presence

of food, variation in pH across GI tract (pH 1–8),

digestive enzymes, intestinal flora, and epithelial

transport (Joshi et al. 2007).

Generally when nutrients are absorbed from the

gastrointestinal tract, primarily through the small

intestines, they immediately enter the portal circula-

tion and undergo the hepatic first-pass metabolism

(Panyam et al. 2002). For many drugs, bypassing the

first-pass metabolism is desired to reduce the clear-

ance of the drug from the bloodstream. However, in

normal condition, this route would mimic the physi-

ological pathway of endogenous insulin produced

from the pancreas (Desai et al. 1996). Nearly half of

the insulin entering portal vein from pancreas is

inactivated in the first passage through liver. Thus,

liver is exposed to a much higher concentration (4- to

8-folds) of insulin than the other tissues (Tripathi

2003). Therefore, while treating pathological condi-

tion i.e., diabetes mellitus via oral route, it is a

prerequisite to design such carrier which could bypass

the hepatic first-pass effect, so that it could increase

oral uptake of insulin or reduce the dose of insulin, as

well as provide the basis for a sustained release insulin

formulation that may provide basal insulin levels for

longer period of time.

Lymphatic absorption is one of the approaches

which enhance oral bioavailability as it bypasses the

hepatic first-pass metabolism (Paliwal et al. 2009).

Targeting of orally delivered peptide and protein drugs

to the lymphatic circulation might be achieved through

one of the following three pathways: paracellular

route, transcellular route, and the gut-associated

lymphoid tissue (GALT) (Mahato et al. 2003).

Absorption through the paracellular route involves

the use of absorption enhancers, which act to ‘‘open

up’’ the tight junctions, thus increasing the permeabil-

ity of hydrophilic macromolecules or macromolecular

conjugates. The higher porosity of lymphatic capil-

laries over blood vessel endothelium directs the

macromolecules to lymphatic circulation. The low

surface area of the paracellular region, however,

limits its applicability because of the possible safety

issues resulting from the long-term use of absorption

enhancers. The transcellular mechanism, on the other

hand, uses the intestinal lipid transport system,

whereby the administered triglycerides are digested

into constituent fatty acids, which are taken up by the

enterocytes, reesterified to triglycerides, and absorbed

into the circulation as chylomicrons. This usually

involves coadministration of lipid-based vehicles, and

the potential drug candidates for this mechanism of

absorption are the lipophilic compounds with log

P [ 5 and triglyceride solubility [50 mg/mL.

GALT consists of lymphoid follicles arranged

singly or in clusters to form distinct structures called

Peyer’s patches. The high concentrations of lymphatic

nodes in the Peyer’s patches toward the lower ileum

have been the subject of much investigation for

targeting (O’Driscoll 2002). A molecular mechanism

that specifically directs solute uptake to lymph over

blood would be valuable in enhancing the oral

absorption of peptide and protein drugs (Granger

et al. 1980). Antigens, particulate carriers, bacteria,

viruses, and protozoa are transported into Peyer’s

patches by specialized epithelial cells known as M

cells. Thus, M cells-targeted oral vaccine and proteins

delivery systems hold promise to improve their oral

efficacy (David and Alan 2004). For proteins/peptide

delivery, nanoparticles offer an attractive possibility,

Page 2 of 14 J Nanopart Res (2012) 14:1219

123

some are targeting M cells (specific targeting) while

others are targeting all intestinal cells, i.e., enterocytes

(non-specific targeting) (Deutel et al. 2008; Jain and

Jain 2008). Nanoparticles have been employed for oral

drug carrier with objectives of improvement of bio-

availability of drug with poor absorption characteris-

tics, delivery of antigens to the GALT, for controlled or

sustained release of drug, reduction of gastrointestinal

mucosa irritation caused by drugs, and for assurance of

stability of drugs in the gastrointestinal tract (Rieux

et al. 2006). Numerous studies have demonstrated the

ability of lectins to bind with intestinal mucosa and their

efficiency for enhancing intestinal uptake of orally

administered particles (Lehr et al.1992; Irache et al.

1996; Ezpeleta et al. 1999; Montisci et al. 2001; Kim

et al. 2005; Mo and Lim 2005; Keegan et al. 2006;

Wood et al. 2008; Kadiyala et al. 2009; Bies et al. 2004).

Therefore, lectins can be grafted onto the surface of

drug carrier and mediate an adhesive interaction

between the carrier and the biological surface (Clark

et al. 2000; Harmony and Cordes 1975). The Con A is a

lectin protein originally extracted from the jack-bean,

Canavalia ensiformis. It binds specifically to non-

reducing terminal alpha-fucosyl groups present in

Peyer’s patches. For oral delivery, Con A is a preferred

candidate because of not only its relatively good

resistance to acidic pH and enzymatic degradation,

but also the ubiquitous presence of binding sites along

the intestinal tract; it also showed greater uptake

potential compared to other lectins like wheat germ

agglutinin (Russell-Jones et al. 1999).

PLGA nanoparticles carrier has high stability in

biological fluids, and is able to avoid enzymatic

metabolism than other colloidal carriers, such as

liposomes or lipid vesicles. They possess high drug-

loading capacities, thereby increasing intracellular

delivery of the drug (Pinto Reis et al. 2006). Owing to

their excellent biocompatibility, the biodegradable

polyester called poly(D, L-lactide-co-glycolide)

(PLGA) is the most frequently used biomaterial and

is already commercialized for a variety of drug

delivery systems (blends, films, matrices, micro-

spheres, nanoparticles, pellets, etc.) (Stevanoviae

et al. 2007). Polymeric nanoparticles of this polymer

are used for the delivery of various drugs (antipsy-

chotics, anesthetics, antibiotics, antiparasites, anti-

tumorals, hormones, proteins, etc.) (Verger et al.

1998). The mechanism of release from nanoparticles

has usually been shown to be biphasic, initially by

diffusion through the polymer matrix and later by

diffusion of the therapeutic agent and degradation of

the polymer matrix itself (Tobio et al. 1998; Dhar et al.

2008; Stevanoviae et al. 2007). PLGA copolymers are

degraded in the body by hydrolytic cleavage of the

ester linkage to lactic and glycolic acid. These

monomers are easily metabolized in the body via the

Krebs cycle and eliminated as carbon dioxide and

water (Panagi et al. 2001; Yoo and Park 2004). In

general, the degradation time will be shorter for low

molecular weight, more hydrophilic, and more amor-

phous polymers, and for copolymers with a higher

glycolide content. An exception to this rule is the

copolymer with 50:50 monomers ratio which exhibits

the faster degradation (about two months).

Therefore, the purpose of the present research work

is to develop surface-modified PLGA nanoparticles,

for improving the oral absorption of insulin by

enhancing its lymphatic uptake.

Materials and methods

Materials

Recombinant Human insulin (27.8 IU/mg) was gifted

from Biocon, Bengaluru, India. PLGA (50:50, MW:

6,600) was a generous gift from National Institute of

Immunology (NII), New Delhi, India. FITC was

purchased from Sigma-Aldrich, MO, USA. Polyvinyl

alcohol (PVA, average Mw 30–70 kDa, 88 % hydro-

lysis), Con A, and dialysis bags (MW cut off

12–14 kDa) were purchased from Himedia, India.

All other chemicals were of analytical grade.

Preparation of insulin-loaded nanoparticles

NPs were prepared using double emulsion solvent

evaporation method (Lamprecht et al. 2001). Primary

emulsion was first prepared by emulsifying 50 mL of

1 % PLGA (50:50) in dichloromethane (DCM) with

aqueous phase consisting of insulin solution (5 mg/

mL) in 0.01 N HCl, and later this primary emulsion

was re-emulsified with aqueous phase consisting of

stabilizer polyvinyl alcohol (PVA, 1 % w/v) solution

using probe sonicator (Lark Innovative Teknowledge,

India) for 60 s. The double emulsion was subjected to

magnetic stirring at 100 rpm for 3 h for complete

evaporation of DCM. The formed NPs were

J Nanopart Res (2012) 14:1219 Page 3 of 14

123

centrifuged at 58,800 9g for 15 min to remove PVA.

Then NPs were lyophilized (Heto Drywiner, Den-

mark) at -46 �C, 0.001 atm for 24 h with 5 % w/w

mannitol which was used as cryoprotectant (Yin et al.

2006).

Preparation of Con A-conjugated nanoparticles

The conjugation was done using the method reported

by Mo and Lim 2005 and Russell-Jones et al. 1999.

The lyophilized NPs were activated by 0.1 M EDAC

in the presence of N-hydroxysuccinimide (NHS)

(0.7 M) and stirred for 2 h at room temperature using

magnetic stirrer. Excess reaction medium was

removed by washing with 50 mL PBS (pH 7.4). The

activated NPs dispersion was added dropwise into

centrifuge tube containing Con A under continuous

vortexing. The reaction was allowed to proceed

overnight and excess coupling sites were blocked by

incubating NPs dispersion with 0.5 mL of glycine

solution (200 mg/mL) for 0.5 h at ambient condition.

The preparation (Con A NPs) was centrifuged at

58,800 9g and washed with bicarbonate buffer (pH

9.5) and resuspended in deionised water and

lyophilized.

Characterization of nanoparticles

FTIR Study

Fourier transform infrared spectrophotometry (FTIR

Spectrometer, BRUKER IFS-55, Switzerland) was

used to study the interaction between insulin and

PLGA as well as to confirm the conjugation of Con A.

The IR spectra of NPs, Con A NPs, insulin, and PLGA

were obtained by the KBr method.

Conjugation efficiency of Con A

Accurately weighed 10 mg lyophilized Con A NPs

were dissolved in 2 mL dimethylsulfoxide and incu-

bated for 5 h at room temperature (25 ± 2 �C) with

10 mL of 0.05 N NaOH/0.5 % Sodium dodecyl

Sulfate (SDS). The resultant solution (1 mL) was

mixed with an equal volume of Bicinchonic acid

(BCA) assay working solution at 60 �C for 1 h and

assayed at 562 nm using UV-spectrophotometer

(Cintra-10, Japan).

Conjugation efficiency %

¼ Wt: of protein ðmcg Con AÞWt: of lyophilized nanoparticles (mg)

� 100

ð1Þ

Shape and surface morphology

The samples for scanning electron microscopy (SEM)

were prepared by lightly sprinkling the lyophilized

NPs and Con A NPs on a double adhesive tape

separately, which was stuck on an aluminum stub. The

stubs were then coated with gold to a thickness of

about 300 A using a sputter coater. All samples were

examined under a scanning electron microscope (Leo

435 VP, Variable press, SEM Co-operation, Leica) at

an acceleration voltage of 30 kV.

Particle size, Zeta potential, and Polydispersity

Index (PDI)

The NPs and Con A NPs samples were diluted 1:9 w/v

with deionized water and analyzed for particle size,

zeta potential, and PDI by photon correlation spec-

troscopy using a Zetasizer (DTS Ver. 4.10, Malvern

Instruments, England).

Entrapment efficiency

Entrapment efficiency of NPs and Con A NPs was

determined using the method described by Xiongliang

et al. 2006. In brief, freshly prepared suspension

NPs/Con A NPs (equivalent to 3.6 mg of insulin) was

centrifuged for 1 h at 4 �C at 58,800 9g (Ultracentri-

fuge, HEMLA Lab. Germany) to separate the un-

entrapped insulin. The supernatant was removed; NPs

and Con A NPs sediments were resuspended in water

and centrifuged twice under the same conditions.

Acetonitrile (200 lL) was added for extraction of

insulin from the sediments and mixture was vortexed

for 5 min. Then, 800 lL of 0.01 M HCl was added

and vortexed for 2 min. After centrifugation at

58,800 9g at 4 �C for 20 min, supernatant was

collected from both the preparations and analyzed

for insulin using RP-HPLC analysis of insulin (SPD-

M20A 230, Shimadzu corporation Kyoto, Japan, Luna

C18 column, 25004.60 mm). The mobile phase con-

sisted of a premixed isocratic mixture of 0.2 M sodium

sulfate anhydrous solution adjusted to pH 2.3 with

phosphoric acid and acetonitrile (73:27, v/v). It was


123

freshly prepared using double-distilled deionized

water, filtered through a 0.22 lm membrane filter.

The injection volume for samples and standards was

20 lL and eluted at a flow rate of 0.8 mL min-1 at

40 �C. The eluent was monitored at 214 nm

(AUFS = 1).

In vitro release study

The in vitro drug release study of NPs and Con A NPs

was performed according to the scheme reported by

Souder and Ellenbogen 1995. Simulated gastric fluid

(SGF; pH 1.2), simulated intestinal fluid (SIF; pH 6.8),

and simulated intestinal fluid (SIF; pH 7.4) were used

as release media sequentially. The NPs and Con A NPs

suspension equivalent to 2 mg of insulin were placed in

the dialysis bags (MW cut off 12–14 kDa, Himedia,

India) separately. They were placed in SGF pH 1.2

(50 mL) at 37 ± 1 �C with continuous stirring with

the help of magnetic stirrer. The samples (0.5 mL)

were withdrawn at 0.5, 1.0, and 2.0 h in SGF (pH 1.2).

Then, the SGF medium was replaced with SIF (pH 6.8)

and study was performed for next 4 h and subsequently

the medium was replaced by SIF (pH 7.4) and study

was performed up to 24 h. The samples (0.5 mL) were

withdrawn at scheduled time interval and withdrawn

samples were replaced with respective fresh medium

and analyzed for drug content using RP-HPLC method

for insulin as described previously. The in vitro drug

release study was repeated at least three times.

Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE)

Polyacrylamide gel electrophoresis was performed to

compare the electrophoretic mobility of insulin before

and after nanoparticles formulation. In addition, the

NPs and Con A NPs were centrifuged at 58,800 9g

15 min to separate insulin from the pelleted-particle

fraction from the soluble supernatant. The pellet and

supernatant were analyzed using SDS-PAGE to deter-

mine relative amounts of insulin associated with each

fraction (Mrisko-Liversidge et al. 2004). For electro-

phoresis, samples were boiled for 5 min in the presence

of sodium dodecyl sulfate and b-mercaptoethanol.

An aliquot of the samples, equivalent to 10–100 lg of

insulin, was electrophoresed on a 16 % SDS poly-

acrylamide gel for 1 h at 200 V. Insulin was visualized

using silver nitrate staining which has a detection limit

of 1–5 ng protein per band. Molecular weight protein

standards were used to compare the electrophoretic

mobility of NPs and unprocessed insulin. The exper-

iment was repeated at least three times.

Circular dichroism study (CD spectrum)

The secondary structure of insulin is necessary for its

bioactivity; therefore, its secondary structure integrity

was analyzed by CD Spectrum. The extracted insulin

with the aid of acetonitrile as described previously

from Con A NPs dissolved in isotonic PBS buffer was

analyzed for its integrity by CD spectra (Jasco, J-815

Spectropolarimeter, UK) at room temperature with

scanning speed of 50 nm/min. The spectra of insulin

samples extracted from NPs and Con A NPs with

concentrations about 10 mM were compared with

pure insulin.

Ex vivo study

Ex vivo and In vivo studies were performed with the

permission of Animal Ethical Committee, Dr. H. S.

Gour University, Sagar, MP (Reg. No. 379/01/ab/

CPCSEA; Per. No. 37).

Intestinal transport study

A method reported by Barr and Riegelman (1970) was

used with modification to assess the intestinal trans-

port of drug (Barr and Riegelman 1970). At first, a

section of small intestine, consisting mainly of ileac

region (about 5 cm) was isolated from a male rat and

washed with Krebs–Ringer bicarbonate solution (pH

7.4). A tube was inserted in one side of the everted

intestine and both the sides were tied with thread and

2 mL of Krebs–Ringer bicarbonate solution was

poured through the hypodermic needle in the tube.

The intestine was placed in a 10 mL medium (Krebs–

Ringer bicarbonate solution) containing NPs formu-

lation (equivalent to 2 mg insulin). The medium was

saturated with O2 and CO2 mixture by slow bubbling

the medium with mixture of gases (95 % O2 and 5 %

CO2) at 37 �C. This set up was done in triplicate for

pure insulin, NPs, and Con A NPs. The samples were

withdrawn periodically from inside of the intestine

and estimated for insulin content using RP-HPLC

method (Xiongliang et al. 2006).


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In vivo study

Histopathological study

Histopathological study was performed to visualize

the localization of the carrier system in Peyer’s

patches and enterocytes. FITC was loaded into NPs

and Con A NPs to yield 2.0 mg FITC/mL of NPs

suspension and it was administered by oral gavage to

three groups (n = 3) of Wistar rats. Group 1 served as

control, while Group 2 and 3 were administered FITC-

loaded NP and FITC-loaded Con A NP (without

insulin), respectively. The rats were sacrificed after

4 h administration of the formulations and the intes-

tinal segment containing Peyer’s patches along with

enterocytes were removed and cut into pieces, washed

with Ringer’s solution, blotted and wiped with tissue

paper. The pieces were fixed in modified Carnay’s

fluid for 3–4 h. The microtomy was performed and

ribbon of sections (thickness 5 lm) was fixed on slides

using egg albumin solution as fixative. Then, the slides

were kept undisturbed for 1–2 days and photographs

were taken using fluorescence microscope (Nikon

Eclips E 600 microscope equipped with video camera

with ECD, software used: Image Proplus).

Blood glucose level (BGL)

Wistar rats weighing 200–250 gm were divided into

four groups (n = 6), housed in cages under controlled

temperature and humidity, and fed a laboratory animal

standard diet and provided tap water ad libitum.

Lighting was on a 12 h on/12 h off cycle and all

animals were induced diabetes with intravenous

administration of streptozotocin (50 mg/Kg) except

group 1 which served as healthy control. After

2 weeks, rats with fasting BGLs above 250 mg/dL

were randomly grouped (n = 6) and used for exper-

iments. These rats were fasted 12 h before the

experiment, but were allowed water ad libitum. Group

2 consisted of untreated diabetic rats (control 2), group

3 was administered marketed insulin preparation

(2.5 IU/kg) by subcutaneous route (control 3), group

4 was administered oral insulin suspension (20 IU/kg)

intragastrically through a stainless steel orogastric

tube, similarly group 5 was administered NPs orally

and group 6 with Con A NPs equivalent to 20 IU/kg in

all groups. The formulations were administered at 0 h

i.e., at start of experiment. Blood samples (about

0.2 mL) were collected from retro orbital plexus at

definite time intervals. BGL of all animals was

monitored periodically using glucometer (Gluco-

trend). During experimentation, standard diet was

given to all the group animals at 4th h and 12th h. The

experiment was repeated at least three times.

Statistical analysis

Data are expressed as mean ± standard deviation

(SD) and statistical analysis was carried out employ-

ing the ANOVA test using the software PRISM

(Graph Pad). A value of p \ 0.05 was considered to be

statistically significant.

Results

NPs were prepared by double emulsion solvent

evaporation method (Lamprecht et al. 2001). Then

NPs were conjugated with Con A using carbodiimide

reaction, i.e., coupling of carboxylic group of PLGA

and amine group of Con A (Fig. 1). FTIR of NPs

shows bands at 1,402.7 and 1,087.1 cm-1, whereas the

FTIR of Con A NPs, shows band at 1,747.0 and

1,650.8 cm-1 (Fig. 2A, B) (Sarmento et al. 2006).

Further, the conjugation efficiency of Con A to

NPs was analyzed using BCA Assay, which was

Fig. 1 Schematic representation of conjugation process


123

found to be 16.13 ± 1.24 lg/mg of NPs (Table 1),

(mean ± SD (n = 6), p \ 0.05).

The prepared NPs and Con A NPs were evaluated

for the shape and surface morphology by scanning

electron microscopy (SEM). SEM study showed

spherical shape with smooth surfaces without any

noticeable pinholes or cracks (Fig. 3).

The average particle size of NPs was found to be

256.30 ± 9.11 nm which is higher as reported by

Cui et al. 2007 which was further increased to

279.1 ± 11.5 nm on coupling with Con A at the

surface of NPs. Further, the NPs were assessed for zeta

potential. The surface of NPs is negatively charged

which was found to be -35.2 ± 1.2 mV and changed

on conjugation to -27.2 ± 1.3 mV. The PDI of NPs

and Con A NPs were found to be 0.129 and 0.137,

respectively. The entrapment efficiency of NPs was

found to be 80.8 ± 3.7 % which was decreased to

72.9 ± 4.3 % as on coupling of Con A at the surface

of NPs which is significantly higher in compare to

that observed by Emami et al. 2009 (Table 1),

(mean ± SD (n = 6), p \ 0.05).

In vitro release study was conducted with NPs and

Con A NPs. In initial 2 h, the drug release from NPs

and Con A NPs in SGF (pH 1.2) was rapid and found to

be 10.6 ± 1.1 and 9.3 ± 1.3 %, respectively; similar

type of burst release was also observed by Emami et al.

2009. In the next 4 h in SIF (pH 6.8) it was found to be

19.4 ± 2.1 and 16.3 ± 1.5 % for NPs and Con A NPs,

respectively. Up to 24 h sustained drug release was

observed reaching a release of 62.3 ± 3.2 % for NPs

and 58.1 ± 2.9 % for Con A NPs in SIF (pH 6.8)

(Fig. 4) (mean ± SD (n = 3), p \ 0.05).

The samples of insulin, supernatant insulin, pellet

of NPs and Con A NPs were run in SDS, the bands of

all the samples were obtained at the equal distance,

i.e., parallel to 5 Kd of the molecular marker (Fig. 5).

Differential scanning colorimetry indicates the endo-

thermic peak of pure insulin at about 70 �C. This

endothermic peak diminishes in the NPs and Con A

NPs at about 50 �C (Fig. 5). The CD spectrum of

insulin in PBS (pH 7.4) shows two minima at 210 and

224 nm. CD spectrum of insulin released from NPs

and Con A NPs shows negligible deviation of the

Fig. 2 FTIR spectra of

A insulin-loaded PLGA

nanoparticles, B insulin-

loaded PLGA nanoparticle

conjugated with Con A (Con

A NPs), C plain insulin, and

D PLGA

Table 1 Optimized parameters of developed formulations

Formulations Particle size

(nm)

Zeta potential

(mV)

PDI %EE Insulin content per 100 mg

nanoparticle

Conjugation efficiency

(lg/mg)

Plain NPs 256.3 ± 9.1 -35.2 ± 5.2 0.259 80.8 ± 3.7 4.04 mg NA

Con A NPs 279.1 ± 11.5 -27.2 ± 1.3 0.347 72.9 ± 4.3 3.64 mg 16.1 ± 1.2

Mean ± SD (n = 6), p \ 0.05


123

negative maxima at about 220 and 223 nm, respec-

tively (Fig. 6).

Histopathological study was performed to visualize

the localization of the carrier along the GI tract. It was

observed with fluorescence microscope, that the group

1 and 4 control section showed intestinal mucosa,

which is characterized by numerous villi with apical

part facing the intestinal lumen. FITC-loaded NPs

were administered to group 2, and were observed in

the lumen of intestine next to apical pole of the villi.

When further focused fluorescent points could be

observed in the connective axis of villi representing

probably FITC-loaded plain NP inside vascular struc-

tures. The FITC-loaded Con A NPs were seen in the

domes of Peyer’s patches more, as compared to

enterocytes situated all along the axis of the villi

(Fig. 7).

Ex vivo study revealed that approx 0.99 ± 0.05,

48 ± 0.9, and 77 ± 1.4 % of insulin was absorbed

across intestinal mucosa from plain insulin suspen-

sion, NPs, and Con A NPs, respectively (Fig. 8),

(mean ± SD (n = 3), p \ 0.05).

On subcutaneous administration of marketed prep-

aration (2.5 IU/Kg) to group 3, the BGL reached

150.13 ± 1.13 mg/dL from 253.66 ± 1.91 after

30 min of administration, this effect prolonged till

4 h of similarly rapid reduction of BGL was reported

after SC administration of insulin by Cui et al. 2007.

The BGL reached 62.17 ± 2.40 mg/dL at 4th h,

which showed peak hypoglycemic effect of subcuta-

neously administered insulin and then the BGL

increases up to initial value (fasting) i.e., 258.66 ±

1.65 mg/dL after 24 h. There was no significant

reduction of BGL on oral administration of insulin

Fig. 3 SEM photomicrographs of A plain nanoparticles and B Con A-conjugated nanoparticles (Con A NPs)

Fig. 4 In vitro drug release

profile from drug-loaded

nanoparticles and CON

A-conjugated nanoparticles

(Con A NPs) at various

gastrointestinal pH. NPsplain insulin loaded

nanoparticles. Con A NpsConcanavalin A-conjugated

insulin loaded nanoparticles.

Mean ± SD (n = 3),

p \ 0.05


123

to group 4 animals. The BGL reduced from

248.17 ± 1.33 to 237.50 ± 1.25 mg/dL within 4 h

in group 5 (oral delivery of NPs), the BGL further

decreased from 295.66 ± 1.73 to 243.33 ± 2.13 mg/

dL in the next 8 h post-feed, depicting the reduction in

BGL prolonging for 8 h and this BGL increased to

250.50 ± 2.11 mg/dL at 16 h equivalent to initial

level. NPs fail to maintain normoglycemic state for the

long time. The Con A NPs on oral administration to

animals of group 6 produced hypoglycemic effect

after [ 3 h of administration, i.e., BGL reduced from

260.17 ± 1.34 to 196.33 ± 1.26 mg/dL. The admin-

istered Con A NPs was able to sustain the glycemic

control post-feed, i.e., the blood glucose decreased for

the next 7 h post-feed from 203.50 ± 1.57 to 97.55 ±

1.10 mg/dL, this effect was further sustained to 24 h

with BGL value of 87.50 ± 2.17 mg/dL (Fig. 9),

(mean ± SD (n = 3), p \ 0.05).

Discussion

The NPs were formed as a result of double emulsifi-

cation process. The primary emulsion was w/o type

and consisted of polymeric solution, which acted as oil

phase, whereas insulin solution in 0.1 N HCl served as

aqueous phase. This primary emulsion was re-emul-

sified using surfactant, i.e., 1 % PVA to form the

secondary emulsion, which was of o/w in nature.

The NPs were formed constituting the oily phase in

this secondary emulsion. This was further purified by

centrifugation and washed with PBS solution. The

NPs preparation was then conjugated with Con A to

confer M-cell targeting potential. Con A NPs were

prepared by coupling of carboxylic group of NPs and

amine group of Con A using EDC/NHS as coupling

reagent involving carbodiimide chemistry (Fig. 1).

The amide bond formation between carboxylic group

of PLGA and amine group of Con A was confirmed by

FTIR spectra. As seen from the FTIR of Con A NPs, it

showed band at 1,747.0 and 1,650.8 cm-1 confirming

–CO stretching (carbonyl group) and –NH bend

(amines), respectively; indicating the formation of

amide bond between carboxylic group of PLGA

and Con A (Fig. 2A, B). BCA assay was used for

determining the Con A conjugation efficiency on NPs

(Table 1).

This conjugation at the surface of NPs leads to

increase in average size of NPs and zeta potential of

NPs is reduced. As the SEM photographs showed

spherical shape with smooth surfaces without any

noticeable pinholes or cracks, which was a result of

optimum emulsification (Fig. 3). The average particle

size of NPs increased on conjugation i.e., from

256.3 ± 9.1 to 279.1 ± 11.5 nm on coupling Con A

at the surface of NPs due to the binding of bulky

molecule i.e., Con A. Zeta potential values revealed

that surface of NPs is negatively charged, i.e.,

-35.2 ± 1.2 mV, which is due to the presence of

uncapped end carboxyl groups of the PLGA at the

surface (Stolnik et al., 1995). Thus, PLGA when

conjugated with Con A which is positively charged

moiety, neutralizes some negative charge of carbox-

ylic group of polymer by forming a covalent

bond and thereby reducing the zeta potential to

-27.2 ± 1.3 mV. The PDI values of NPs and Con

A NPs indicated the narrow size distribution of the

prepared formulation (Table 1). The decrease in

entrapment efficiency on coupling could be attributed

to the residual leakage of drug from the NPs during

incubation period employed for coupling of Con A

(Table 1).

In order to evaluate the suitability of nanoparticles

as a delivery system for insulin, an in vitro release

study was conducted with NPs and Con A NPs. In

Fig. 5 SDS-PAGE photomicrograph (Lane M-molecular mar-

ker, Lane 1-Insulin, Lane 2-Supernatant insulin from insulin-

loaded nanoparticles (NPs), Lane 3-Pellet of insulin-loaded

nanoparticles (NPs), Lane 4-Supernatant insulin from Con

A-conjugated nanoparticles (Con A NPs), and Lane 5–Insulin

extracted from pellet of Con A-conjugated nanoparticles (Con A

NPs)


123

initial 2 h, the drug release from NPs and Con A NPs

in SGF (pH 1.2) was rapid due to burst release. Up to

24 h a sustained drug release was observed for NPs

and Con A NPs in SIF (pH 6.8), which is attributed to

polymeric encapsulation of insulin, which controls its

release pattern. The lower drug release was observed

with Con A NPs than NPs due to double barrier effect

exhibited by bulky Con A (Fig. 4). Insulin due to its

peptidal nature is subjected to degradation during its

formulation procedure (Chalasani et al., 2007). There-

fore, it is necessary to determine its structural integrity

which was done by performing SDS-PAGE which

Fig. 6 CD-Spectra of A Human Insulin, B Extracted insulin from plain nanoparticles, and C Con A nanoparticles (Con A NPs)

Fig. 7 Fluorescence Photomicrographs of Intra ileal sections A control section, B FITC-loaded plain nanoparticles, and C FITC-

loaded Con A conjugated nanoparticles


123

indicated the structural stability of the native insulin

and entrapped insulin, as it confirms that there is no

breakage of disulfide bonds between the two A and B

chain of insulin molecule, which is necessary for

drug’s activity (Fig. 5).

Preservation of structural integrity of protein drug

after release is essential for biological efficacy. Thus,

the secondary structure of insulin released from NPs

and Con A NPs were investigated using CD spectra.

CD spectrum of insulin released from NPs and Con

A NPs shows negligible deviation of the negative

maxima at about 220 and 223 nm, respectively. The

secondary structure for insulin (native conformation)

may have been very slightly affected after encapsu-

lation. The possible explanation for such an observa-

tion would be the shear forces which acted during the

formulation process, though not representing denatur-

ation or loss of insulin activity (Fig. 6).

Histopathological studies showed that there was

better uptake of FITC-loaded Con A NP and their

localization in Peyer’s patches in group 3 compared to

group 2 (FITC loaded NPs) (Fig. 7), and the ex vivo

study (Fig. 8) revealed that the higher insulin absorp-

tion from Con A NPs than the non-encapsulated

insulin is due to capacity of the polymeric matrix of

nanoparticles to protect the drug, and better absorption

due to targeted approach. In case of Con A NPs, the

highest drug uptake which was observed in the above-

mentioned studies may be due to increased transcel-

lular uptake of Con A NPs via M cells, as Con A

exhibits affinity for a-L-fucose residues present on M

cells in Peyer’s patches of ileac intestine, and

relatively less negative charge which also increases

interaction with relative electronegative intestinal

epithelium due to sialic acid residues and natural

affinity of Con A ligand for a-L-fucose residues in

Peyer’s patches.

As diabetic rats were selected for the in vivo study

which might have died if continuously fasted, standard

feed was given to all the animals at 4, 12, and 24 h of

study, which raised BGL values at 5th h and 14th h till

2 h post-feed.

On Subcutaneous administration of marketed prep-

aration to group 3, the BGL lowering effect was seen

after 30 min of administration, this effect prolonged

till 4 h. With time it’s hypoglycemic effect decreasesFig. 8 Graph between time and percent insulin transported

across intestinal barrier. Mean ± SD (n = 3), p \ 0.05

Fig. 9 Blood Glucose

Profiles at various time

intervals on oral

administration of differentformulations compared with

subcutaneous administration

of marketed preparation.

Mean ± SD (n = 3),

p \ 0.05


123

and hence the BGL rises to 258.66 ± 1.65 mg/dL

after 24 h as initial value (fasting). The conventional

administration leads to a hypoglycemic response

which is not sustained, in other words there is peak

and troughs in its action.

On oral administration of insulin to group 4

animals, no significant lowering of BGL was observed

due to degradation of insulin in GIT lumen attributed

to different enzymatic processes, pH conditions, and

physical barriers. While in group 5 orally delivered

NPs reduced BGL within the first 4 h, which subse-

quently raised after feed. However, the NPs were able

to sustain reduction in BGL value over next 8 h.

NPs fails to maintain normoglycemic state as the

insulin was released in the intestinal tract and

degraded in situ; however, some of the insulin was

taken up by the passive uptake mechanism and was

transported through portal circulation, and got

exposed to the first-pass effect of liver. On the other

hand, the Con A NPs on oral administration to animals

of group 6 produced hypoglycemic effect after 3 h of

administration, i.e., BGL reduced from 260.17 ± 1.34

to 196.33 ± 1.26 mg/dL, which was due to the gastric

emptying time required by the NPs preparation to

reach intestine. The hypoglycemic effect was sus-

tained for the next 24 h of Con A NPs administra-

tion.This reduction of blood glucose till 24 h of its

administration could be attributed to synergistic effect

of drug encapsulation within polymeric nanocarrier,

which gives the sustained release profile to the

encapsulated insulin and the targeted approach

enhances the lymphatic uptake through M Cells,

which is a energy-dependent transcellular process.

The hydrophobic nature of Con A NPs and the overall

negative charge as well as Con A affinity to sugar

residues, i.e., a-L-fucose present on M cells increase its

uptake efficiency and better pharmacological response

as seen by the BGL values compared to other groups.

The NPs due to their size are transcytosed as such by

the M Cells and delivered inside the lymphatic vessels,

and the encapsulated insulin is released in due course

and reaches the blood circulation without getting

exposed to the liver, which may otherwise degrade

fifty percent of administered dose. Thus, the resulting

Con A NPs has potential to act as oral delivery system,

which could be effective in management of Type 1

diabetes. The present formulation could serve as long-

acting basal dose, which may modulate the fluctua-

tions in sugar levels in diabetes condition.

However, further work is going on in our laboratory

using different targeting ligands with PLGA nanopar-

ticles and combining such a system with enteric

polymers so as to attain more controlled release and

better stability.

Conclusion

In conclusion, the present work resulted in successful

production of Con A NPs with sustained release

profile, better absorption and stability as shown in

in vitro, ex vivo, stability, and in vivo studies. The

increase in absorption of insulin from NPs than the

non-encapsulated insulin is attributed to the capability

of the polymeric matrix to protect the drug and better

distribution leading to absorption. The Con A NPs

showed higher insulin uptake, as compared to NPs due

to its relative high affinity for non-reducing carbohy-

drate residues i.e., fucose present on M cells, which

aids in increased bioavailability and better therapeutic

response for orally administered insulin. Thus, Con A

NPs have the potential for oral insulin delivery in

effective management of Type 1 diabetes condition.

Acknowledgments The authors acknowledge the financial

assistance from All India Council of Technical Education

(AICTE), New Delhi, India. The authors are thankful to Prof.

A.K. Panda, National Institute of Immunology, New Delhi,

India, for generously providing gift sample of PLGA (50:50).

Declaration of interest The authors report no conflicts of

interest. The authors alone are responsible for the content and

writing of the paper.

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