ORIGINAL PAPER

Metformin-Loaded BSA Nanoparticles in Cancer Therapy: A NewPerspective for an Old Antidiabetic Drug

Pinkybel Jose • K. Sundar • C. H. Anjali •

Aswathy Ravindran

� Springer Science+Business Media New York 2014

Abstract Clinical and experimental data suggest that there

is a strong association between type II diabetic mellitus and

pancreatic cancer. The present study focuses on exploring the

anticancer and antidiabetic properties of metformin-loaded

bovine serum albumin nanoparticles (BSA NPs) on (Mia-

PaCa-2) pancreatic carcinoma cell lines. Albumin nanopar-

ticles were synthesized using coacervation method and the

average size of the particles was found to be 97 nm. The

particles were stable and showed a spherical morphology with

narrow size distribution. We investigated the impact of two

stages characterized in type II diabetes mellitus (hyperglyce-

mia and hyperinsulinemia) on the proliferation of MiaPaCa-2

cells and compared the inhibitory effects of bare metformin to

that of MET-BSA NPs. Further, different concentrations of

insulin and glucose were added along with bare metformin,

bare BSA, and metformin encapsulated BSA carrier on Mia-

PaCa-2 cells to check the strong association between type II

diabetes and pancreatic cancer. The results revealed that

MET-BSA NPs showed more toxicity when compared with

drug and carrier individually.

Keywords BSA, MiaPaCa-2 � Hyperglycemia �Hyperinsulinemia � Pancreatic cancer

Introduction

Ground-breaking research is being conducted, the world

over, in the field of nanotechnology [1, 2]. One of the most

critical outputs is the development of new materials on the

nanometer scale [3]. Slow progress in the treatment of

several diseases has resulted in an increased need for a

multidisciplinary methodology termed drug delivery sys-

tems [4, 5]. The compelling features of this approach are

enhanced specificity, in terms of drug targeting and

delivery as well as minimum amount of nanoparticle usage.

Nanoparticles are utilized in drug delivery systems,

because their surface to mass ratio is much larger than that

of other particles [6]. These particles also have an effect on

the following properties: increasing the stability of drugs,

higher payload capacity, prolonged blood circulation times,

reduced toxicity to healthy tissues, and improved antitumor

efficiency [3]. Nanocarrier composed of materials, such as

proteins, polysaccharides, polymers, and liposomes offers a

number of advantages making it an ideal drug delivery

vehicle [7–15].

Serum proteins serve as an apt material for nanoparticle

formation as it is naturally found in the blood, non-toxic,

biodegradable and non-immunogenic properties [16].

Serum albumin is the most abundant protein in the circu-

latory system and is chiefly responsible for the mainte-

nance of blood pH [17]. Bovine serum albumin (BSA) is a

transport protein capable of binding many exogenous and

endogenous drugs reversibly. In this study, BSA was

chosen as the nanocarrier owing to its easy availability and

biocompatible nature [18]. Seven types of medications are

commonly used to treat diabetes: insulin, biguanides (i.e.,

metformin hydrochloride), sulfonylurea’s (e.g., glyburide,

glipizide), meglitinides (i.e., repaglinide), phenylalanine

derivatives (i.e., nateglinide), alphaglucosidase inhibitors

(i.e., acarbose, miglitol), and thiazolidinediones (i.e.,

pioglitazone and rosiglitazone). Metformin hydrochloride,

a biguanide agent for the treatment of type II diabetes is

prescribed to about 120 million people worldwide [19, 20].

P. Jose � K. Sundar � C. H. Anjali � A. Ravindran (&)

Center for Nanotechnology and Advanced Biomaterials

(CeNTAB), School of Chemical and Biotechnology, SASTRA

University, Tirumalaisamudram, Thanjavur 613401, Tamilnadu,

India

e-mail: aswathy85@gmail.com

123

Cell Biochem Biophys

DOI 10.1007/s12013-014-0242-8

Metformin was utilized in this study as it has been proved

to be the only antidiabetic drug that prevents the cardio-

vascular complications of diabetes [21, 22]. Even though

this biguanide is used as the first line therapy for Type II

diabetes mellitus, it may also increase the risk of vitamin

B12 deficiency and folate deficiency. Increase in homo-

cysteine concentrations, an independent risk factor for

cardiovascular disease especially among Type II diabetes

mellitus with metformin has also been reported [23]. Hence

the current study focuses on overcoming the existing

drawbacks of metformin hydrochloride by encapsulating it

in a BSA nanocarrier.

According to the recent reports, pancreas is generally

exposed to substantial hyperinsulinemia for years in patients

with type II diabetes mellitus. This suggests that insulin may

be involved in the association between long-standing dia-

betes and pancreatic cancer [24], and the factors associated

with abnormal glucose metabolism may play an important

role in the pancreatic cancer [25]. Even though the antidia-

betic properties of metformin are well explored, the over-

looking the anticancer properties of metformin are still in its

infancy. Hence in the absence of prior reports, our study aims

to explore the antidiabetic and anticancer properties of

metformin encapsulated BSA NPs. We have mimicked the

different stages of type II diabetes mellitus with various

combinations of insulin and glucose to see the cancer pro-

liferation, and the direct effect of bare and MET-BSA NPs on

the apoptosis of pancreatic cells.

Materials and Methods

BSA (fraction V, minimum 98 %), 25 % glutaraldehyde

solution and mannitol used as a cryoprotectant were pur-

chased from Sigma Aldrich, India. Metformin Hydrochloride

powder used for the experiments was of pharmaceutical grade.

MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-

phenyl)-2-(4-sulfonyl)-2H-tetrazolium) and propidium iodide

were purchased from Sigma–Aldrich. Dulbecco’s modified

Eagle’s medium (DMEM), DMEM without glucose, human

insulin solution, D glucose solution, and fetal bovine serum

(FBS) were supplied by Invitrogen. Human pancreatic cancer

cell line (MiaPaCa-2) was obtained from National Center for

Cell Sciences, Pune, India. Ethanol and acetone were used as

desolvating agents. Phosphate buffered saline (PBS) was

prepared from NaH2PO4 and Na2HPO4. Distilled deionized

water was used for performing experiments.

Maintenance of Cell Lines

Pancreatic cancer cells (MiaPaCa-2) were cultured in

DMEM containing 10 % FBS, 100 unit/mL penicillin G,

and 100 lg/mL streptomycin at 37 �C in a 5 % CO2

humidified atmosphere.

Preparation of Bare and Metformin-Loaded BSA

Nanoparticles

BSA NPs were prepared by coacervation method [26].

BSA was first dissolved in deionized water and incubated

at room temperature. The pH of the solution was adjusted

to 8.0, and ethanol was added drop wise at a controlled rate

under stirring conditions. The coacervate so formed was

cross-linked with glutaraldehyde and kept for stirring for

24 h to form stable nanoparticles. The resulting nanopar-

ticles were centrifuged, and pellets obtained were freeze

dried out using mannitol as a cryoprotectant. Lyophiliza-

tion was done to get fine powder of BSA NPs. Metformin-

loaded BSANPs were also prepared by the same method in

which metformin was incubated with BSA for 24 h before

stirring. Various parameters were altered in order to

understand the factors affecting the properties of the

nanoparticles prepared. Optimization of BSA NPs was

done by varying the pH of the solution, % of glutaralde-

hyde added for cross linking, and rate of ethanol added for

promoting coacervation. Best parameters were selected to

synthesize BSA NPs of minimum size [27].

Physicochemical Characterization of the Particles

Following Encapsulation

The hydrodynamic size and zeta potential of nanoparticles

were determined by Malvern Zeta sizer 3000HS (Malvern

Instruments Ltd., UK). The surface morphology of the drug

encapsulated and bare nanoparticles were analyzed using

Field Emission Scanning Electron Microscopy (JEOL,

JSM-6360, scanning electron microscope, Japan) at 15 kV.

All fluorescence measurements were recorded with LS 55

Fluorescence Spectrometer (Perkin Elmer) to elucidate the

nature of interaction of drug and the carrier. FT-IR spectra

of the drug-loaded and unloaded particles were examined

using KBr pellet (1 % (w/w) of product in KBr) with a

resolution of 4 cm-1 on Perkin Elmer Spectrum RXI

Fourier Transform Infrared spectrophotometer.

Drug Encapsulation Efficiency and Drug Release

Kinetics

Following synthesis, the drug encapsulation efficiency of

the carrier was estimated by the ratio of measured and

initial amount of metformin encapsulated in nanoparticles.

For drug release kinetics, the MET-BSA NPs suspension

containing 10 mg of metformin was kept for dialysis by

immersing it into 20 mL of PBS at pH 7.4. Samples were

stirred at 37 �C, and PBS was replaced with fresh medium

Cell Biochem Biophys

123

at definite time intervals. The released drug was quantified

at 232 nm using UV–Vis spectrophotometer.

Cell Proliferation Assay

Anticancer Property of Metformin on Cancer Cells

For the in vitro studies, 10,000 cells/well were seeded in

96-well plates in quadruplicate and treated with different

concentrations of bare metformin, bare BSA and metfor-

min-loaded BSA NPs. MTS assay was performed, and the

number of living cells following the respective treatments

was quantified by measuring the absorbance of formazan,

using 1420-040 Victor3 Multilabel Counter (Perkin Elmer,

USA) at 490 nm [28, 29].

The surviving fraction of cells was calculated by the

formula:

% of surviving cells ¼ OD of test sample

OD of control� 100

The control sample reading was obtained from the

untreated wells; the reading was a mean of four wells. All

treated wells were assayed at least in quadruplicate, and

results were expressed as mean percent surviving cells

[30].

Measurement of Reactive Oxygen Species

The fluorescent dye 6-carboxy 20,70-dichlorodihydro-

fluorescein diacetate was used to determine reactive oxy-

gen species (ROS) which is a natural byproduct of normal

metabolism of oxygen. The cultured cells were treated with

5 lM DCF dye in PBS, and change in fluorescence was

recorded at 490 nm using Tecan fluorescence plate reader

[31].

Hemocompatibility of the Drug-Loaded Nanoparticles

The hemocompatibility of the metformin-loaded BSA NPs

can be studied using hemolysis test. Blood was drawn from

a healthy volunteer and transferred to the tube containing

anticoagulant solution of EDTA and incubated with 0.9 %

physiological saline for 37 �C. MET-BSA NPs were added

to the diluted blood and kept for incubation at 37 �C for

60 min. Blood with distilled water was taken as positive

control, and 0.9 % saline with blood was taken as negative

control. Samples were centrifuged at 3,000 rpm for 5 min

after incubation.

Cell Uptake Studies

The cellular internalization of metformin-loaded BSA NPs

by cancer cells were evaluated by fluorescent microscopy

using propidium iodide staining. Propidium iodide is a polar

dye and highly soluble in water [32]. The cells were seeded in

a density of 10,000 cells/well in a 96-well plate. After 24 h

of cell attachment, the media was removed, and the wells

were carefully washed with PBS. MET-BSA NPs of different

concentration (in triplicates) was added along with media

into these wells and incubated for 24 h. Later, propidium

iodide was added to each well. After 15–20 min, cells were

washed with PBS, and cells were fixed in 5 % paraformal-

dehyde followed by a final PBS wash. The cells were then

mounted on to glass slides with distyrene plasticizer xylene

as the mountant, and then dead cells were viewed under the

fluorescence microscope. The extent of dead cells obtained

after treatment with bare metformin was compared with BSA

entrapped metformin.

Checking the Impact of Glucose and Insulin on Cancer

Cells

The impact of glucose and insulin was checked on Mia-

PaCa-2 cancer cell line since cancer promotes hypergly-

cemia and hyperinsulinemia, characterized by type II

diabetes [33, 34]. In this study, pancreatic cancer cells

(MiaPaCa-2) were chosen at a density of 10,000 cells per

well and cultured in DMEM with 10 % FBS. Different

concentrations of insulin and glucose were added to

DMEM without glucose to mimic the different stages of

type II diabetes (normal, prediabetic, overt diabetic, and

late diabetic) with 0.5 % to replace the original DMEM

after 24 h. 20 lL of MTS solution was added to each well

after 72 h of incubation [35]. The absorbance was mea-

sured using 1420-040 Victor3 Multilabel Counter (Perkin

Elmer, USA) at 490 nm after incubating the plate in the

dark conditions for 2 h.

Results and Discussion

Preparation and Characterization of Nanoparticles

In order to understand the factors affecting the properties of

NP prepared from the coacervation procedure, series of

experiments were performed by controlling the BSA con-

centration, the pH of coacervation medium, and the non-

solvent/water ratio during NP formation [36, 37]. According

to Weber et al., and Langer et al., the amount and rate of

addition of the solvent, pH, and the concentration of glu-

taraldehyde added, critically affect the coacervation process.

The concentration of BSA was varied from 10–30 mg/mL

under different pH conditions (4, 8, and 10). As shown in

Fig. 1, a gradual reduction in NP size was observed when the

BSA concentration was 20 mg/mL. The polydispersity

index of BSA nanoparticles was recorded to be 0.041 which

Cell Biochem Biophys

123

indicated a uniform size distribution of the particles. The

average hydrodynamic size of the particles varied from 97 to

120 nm with a zeta potential value of 32.5 mV (Fig. 1).

Following encapsulation with metformin an increase in

hydrodynamic size to 127 nm and zeta potential value of

39.3 mV was recorded. Since metformin is insoluble in

acetone, ethanol was used to promote coacervation, and our

results revealed that the amount of ethanol added influenced

the particle size. Narrow size distribution of the particles was

obtained when the rate of addition of ethanol was 0.8–1 mL/

min. Glutaraldehyde was used as a cross linker since it

exhibited high reactivity toward the amino groups of albu-

min nanoparticles [38, 39]. Since cross linking was not seen

at low concentrations of glutaraldehyde, 100 lL of 8 %

glutaraldehyde was optimum to form stable protein nano-

particles. By varying the protein and drug ratio, particles

were synthesized, and less size was obtained when 20 mg of

BSA, and 15 mg of metformin was used. Thus after opti-

mizing the parameters, MET-BSA NPs were synthesized

with pH 8, the rate of ethanol added at 0.8–1 mL/min, 8 %

glutaraldehyde, and 10:20 mg of drug: protein ratio.

The scanning electron microscopic analysis revealed

spherical morphology of the BSA NPs before and after

encapsulation with the drug metformin as shown in Fig. 2.

These results were very well in accordance with results

obtained after hydro dynamic size and zeta potential

measurements. In order to understand the nature of binding

of the drug to the BSA NPs fluorescence spectroscopy was

carried out. There are mainly two types of fluorophores in

BSA namely, tryptophan and tyrosine residues [40, 41].

When excited at 280 nm, both tryptophan and tyrosine

residues exhibited fluorescence quenching whereas at

293 nm, only tryptophan residues excited [42]. The sig-

nificant difference in the quenching of serum albumin

fluorescence at 280 and 293 nm showed that both residues

play an important role in the molecular interaction of BSA

and metformin (Fig. 3).

FT-IR analysis of nanoparticles in Fig. 4 showed peaks

at 935 and 800 cm-1 corresponding to the N–H wagging of

metformin and BSA, respectively. MET-BSA NPs were

observed with the spectra at 901 and 879 cm-1. This

clearly shows the presence of metformin is encapsulated by

BSA NPs.

Drug Encapsulation Efficiency and Drug Release

Kinetics of the Nanoparticles

Various concentrations of drug were taken and checked for

the entrapment efficiency. The concentration of the drug

was varied from 5–20 mg of BSA. Maximum entrapment

efficiency (92 %) was found at 10:20 mg of drug:BSA.

Encapsulation efficiency decreases as drug concentration

increases. The drug release profiles of metformin-loaded

nanoparticles were also investigated. Metformin-loaded

nanoparticles were tested for drug release at 37 �C in PBS

at pH 7.4. Within 24 h, 50 % of the drug released and rest

of the drug got released within 72 h. The release profile

showed that the pharmacologically active drug was

released in a slow and sustained manner (Fig. 5).

Fig. 1 Data show the optimization of process parameters

Fig. 2 SEM images of BSA NPs. a Before and b after encapsulation with metformin

Cell Biochem Biophys

123

Cytotoxicity Studies

Cell Proliferation of Cancer Cells by Insulin and Glucose

Epidemiologic evidence suggests that patients with diabe-

tes are at a significantly higher risk of developing many

types of cancers, particularly cancers of the pancreas,

breast, liver, esophagus, and colons [30]. According to the

recent reports, cancer patients with diabetes are predomi-

nantly type II in nature [30, 31]. As Type II diabetes is

characterized by increased glucose and insulin levels, the

effect of these components on the progression of MiaPaCa-

2 cells were studied [43, 44]. Our results revealed that there

was a considerable increase in cancer proliferation with

increase in insulin (0.01–5 lg/mL) and glucose

(1,000–4,000 mg/mL) levels (Fig. 6). The growth of can-

cer cells was accelerated at a high glucose concentration of

4,000 mg/L and insulin concentration of 5 lg/mL These

results were supported by the findings of Han et al. [31]

who reported that high glucose levels promoted cell pro-

liferation through the regulation of expression of glial cell

line-derived neurotropic factor and RET in pancreatic

cancer cells. Hence our studies were consistent with the

above findings [45].

Further in order to check the effect of antidiabetic drug

(metformin) on the growth of pancreatic cancer cells, we

examined the impact of different concentrations (10, 20,

100 lg/mL) of BSA NPs, BSA-loaded and unloaded met-

formin on the proliferation of MiaPaCa2 cells in culture

Fig. 3 Fluorescence quenching

of BSA NPs by metformin at

280 and 293 nm

Fig. 4 Curves show the FT-IR spectra of bare metformin and

MET-BSA NPSFig. 5 Curve shows the in vitro drug release pattern of metformin

from the BSA NPs

Cell Biochem Biophys

123

Fig. 6 Cellular toxicity of

different concentrations of

a insulin, b glucose, and

c combination of glucose and

insulin on MiaPaCa2 cells

Fig. 7 Graph shows the impact of various concentrations of a drug ? carrier, b drug, and c carrier on MiaPaCa2 cells

Cell Biochem Biophys

123

media containing 0.1 and 5 lg/mL of insulin and 2,000 and

4,000 mg/mL of glucose, respectively. We found that

metformin inhibited MiaPaCa2 cell proliferation in all

studied combinations of glucose and insulin tested whereas

BSA NPs exhibited little or no toxicity (Fig. 7). Interest-

ingly, MET-BSA NPs showed a dose-dependent inhibition

in proliferation in all combinations of glucose and insulin

tested. However, BSA alone which acted as a control did

not exhibit any growth inhibition with respect to control

following cell viability assay [44].

Anticancer Properties of Metformin-Loaded BSA

Nanoparticles

Cell proliferation assay was done to estimate the cell via-

bility in cancer cells seeded with the drug, carrier, and drug

with carrier. Cell viability decreases with increasing the

concentrations of metformin, BSA NPs, and MET-BSA

NPs. The MET-BSA NPs showed significantly higher

toxicity than the bare drug and carrier. Metformin activates

AMPK pathway, a major sensor for the energetic status of

the cell, which has been proposed as a promising thera-

peutic target in cancer [46]. One possible reason why

MET-BSA NPs show higher toxicity may be due to the

enhanced signaling of AMPK pathway by the sustained

release of metformin from MET-BSA NPs. Figure 8a

shows the cell viability of the drug, BSA NPs and MET-

BSANPs.

Possible Mechanism—Reactive Oxygen Species (ROS)

ROS-free radicals production is one of the primary

mechanisms of nanoparticle toxicity. ROS increase is

thought to play an important role in maintaining cancer

phenotype due to their stimulating effects on cell growth

and proliferation, genetic instability and senescence eva-

sion. An increased ROS in cancer cells is often consid-

ered as an adverse factor. Higher levels of ROS can also

cause cellular damage, depending on the levels and

duration of ROS stress. [47]. In the present study, we

incubated different concentrations of the nano formula-

tions with the cell lines. After an incubation time of about

48 h, the cells were analyzed using a fluorescent plate

reader Tecan. The ROS produced in the cells incubated

with the test samples were calculated with respect to the

blank cells. A double-fold increase in production of ROS

was noted in the case of MET-BSA NPs when compared

to equivalent concentrations of metformin and BSA NPs.

Although the ROS production is not the primary cause for

the cytotoxicity, a significant amount contributes to the

toxic effects caused to the cells. The fold increase in ROS

production with respect to the concentrations of the drug

can be seen in Fig. 8b.

Fig. 8 Graph a shows viability

of pancreatic carcinoma cells

(MiaPaCa-2) after 48 h of

exposure to 10, 15, 20, 50, and

100 lg/mL MET-BSA NPs,

metformin and BSA NPs as

determined by MTS assay.

Percent relative viability is

expressed relative to the control.

b The ROS production

following treatment of various

concentrations of drug,

drug ? carrier, and carrier on

MiaPacCa-2 cells

Fig. 9 Blood compatibility with MET-BSA NPs, normal saline

diluted with ant coagulated blood (negative control) and distilled

water with anticoagulated blood (positive control)

Cell Biochem Biophys

123

Haemocompatibility Studies

MET-BSA NPs of different concentrations were incubated

with the blood samples, and the results were observed. Our

results clearly demonstrated that there was no lysis of the

RBC’s in the supernatant in all the incubated samples

which demonstrated that nanoformulation is perfectly

suitable for the circulation in blood. No adverse reactions

were observed between the serum proteins and the surface

of the nanocarrier (Fig. 9). Distilled water which was used

as a positive control produced the complete lysis of the red

blood cells (RBC’s). 0.9 % NaCl (physiological saline)

used as the negative control did not show any hemolysis or

toxicity to the RBC’s hence the percentage of lysis is 0.

The blood samples incubated with the nano formulations

showed results similar to that of saline, and hence it can be

concluded that the formulation was haemocompatible in

nature and did not induce thrombus formation.

Cell Uptake Studies

Internalization of the drug nanoparticles and its effect on

cancer cells can be seen in Fig. 10. The fluorescent marker

propidium iodide (PI) was used to see the dead cells. PI

staining was done on drug, BSANPs and MET-BSANPs

incubated in Mia PaCa-2 cells. Cytotoxic studies of bare

metformin were compared to that of MET-BSANPs. The

amount of dead cells was more in MET-BSANPs when

compared to that of bare metformin, which reveals the

efficacy of the drug-loaded carrier [32].

Conclusion

Metformin-loaded BSA NPs were synthesized using

coacervation method. The as synthesized nanoparticles

were spherical with a uniform size distribution. Pharma-

cological release kinetics of MET-BSA NPs was slow and

in a sustained manner. The cell proliferative toxic effects

exhibited by MET-BSA NPs were significantly high when

compared to bare metformin. Furthermore the production

of more ROS in MET-BSA NPs shows the efficacy of the

drug encapsulated in the carrier in terms of toxicity.

Additionally, the hemolysis assay done on normal human

RBC’s suggests the safe encapsulation of MET-BSA NPs.

The addition of glucose and insulin to MiaPaCa-2 cells

showed a dose-dependent inhibition of proliferation in cells

with MET-BSA NPs. Interestingly, bare BSA NPs did not

exhibit any growth inhibition in cancer cells. The propi-

dium iodide-stained MET-BSA NPs revealed the presence

of more dead cells than bare metformin, which also add to

the aforementioned high toxic effects of MET-BSA NPs.

Prospectively, this study can be used for a better under-

standing of the anticancer properties of metformin.

Fig. 10 Images show the cellular uptake image of MiaPacCa-2 cells after PI staining with metformin (a, b) and MET-BSA NPs (c, d)

Cell Biochem Biophys

123

Acknowledgments Authors would like to acknowledge Depart-

ment of Science and Technology for providing the external funding to

carry out the project entitled, ‘‘Biocompatibility of surface modified

and unmodified Graphene oxide nanoparticles’’ (NO:SR/FT/LS-18/

2012). We would also thank Centre for Nano Technology &

Advanced Biomaterials (CeNTAB) and Centre for Advanced

Research in Indian System of Medicine (CARISM), SASTRA Uni-

versity for providing the facilities to carry out this project.

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