Materials Science and Engineering C 31 (2011) 1305–1312

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Materials Science and Engineering C

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Controlled release of drug from folate-decorated and graphene mediated drugdelivery system: Synthesis, loading efficiency, and drug release response

D. Depan a, J. Shah b, R.D.K. Misra a,⁎a Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette,LA 70504-4130, USAb Global Nanotech — A Nanomaterials Company, Jawahar Nagar, S.V. Road, Goregaon West, Mumbai 400 062, India

⁎ Corresponding author. Tel.: +1 337 482 6430; fax:E-mail address: dmisra@louisiana.edu (R.D.K. Misra)

0928-4931/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.msec.2011.04.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 January 2011Received in revised form 26 March 2011Accepted 4 April 2011Available online 12 April 2011

Keywords:ChitosanGraphene oxideFolic acidDoxorubicinControlled drug release

A novel folate-decorated and graphene mediated drug delivery system was prepared that involves uniquelycombining graphene oxide (GO)with anticancer drug for controlled drug release. The nanocarrier systemwassynthesized by attaching doxorubicin (DOX) to graphene oxide via strong π–π stacking interaction, followedby encapsulation of graphene oxide with folic acid conjugated chitosan. The π–π stacking interaction,simplified as a non-covalent type of functionalization, enables high drug loading and subsequent controlledrelease of the drug. The encapsulated graphene oxide enhanced the stability of the nanocarrier system inaqueous medium because of the hydrophilicity and cationic nature of chitosan. The loading and release ofDOX indicated strong pH dependence and imply hydrogen-bonding interaction between graphene oxide andDOX. The proposed strategy is advantageous in terms of targeted drug delivery and has high potential toaddress the current challenges in drug delivery. Thus, the prepared nanohybrid system offers a novelformulation that combines the unique properties of a biodegradable material, chitosan, and graphene oxidefor biomedical applications.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Recently, more attention has been paid on life threatening disease,cancer, which continues to increase with increasing age of thepopulation and urbanization. Although, the medical science andbiomedical engineering advanced to a significant extent, the thera-peutic development of anti-cancer strategies is often limited byadministration problem of the drugs. Moreover, the malignancy oftumors is detected only at advanced stages when administration ofchemotherapeutic drugs is toxic to healthy cells. In this context, recentattempts have been made explore targeted drug delivery [1] anddetect cancer cells at an early stage are of particular interest [2]. Forefficient drug action, improving the drug loading efficiency is critical indrug carrier research. On the other hand, site-directed drug targeting isalso very important for improving drug efficiency and decreasing thedrug's side effects. Tumor-targeting drug delivery system generallycombines tumor recognition moiety with drug-loaded vesicle [3–6].

Graphene and graphene oxide (GO) is a novel atom-thick two-dimensional graphitic carbon [7], and has attracted significantattention from material researchers, because of favorable physicaland biological properties [8]. For instance, the solubility of GO nano-sheets in water and other polar solvents has led to their consideration

for applications including nano-electronics, sensors, and nanocompo-sites [9]. Although, GO provides large specific surface area for theimmobilization of various biomolecules, the biological applications ofGO remain virtually unexplored. It has been proposed to considerexploring graphene for drug delivery [10–12].

In a recent study, the biocompatibility of graphene was confirmedby examining the growth of fibroblast cells (L-929) [13] and cellularuptake of insoluble drugs [14–16].

Natural biodegradable polymers such as cyclodextrin, polylactideand chitosan (CHI) are viable polymeric materials to enhance thebiocompatibility of the matrix for controlled release of therapeuticmolecules [17–21] and folic acid (FA) is a ligand for targeting cell-membrane and promoting endocytosis via the folate receptor. Further-more, it is a stable and poorly immunogenic chemical with high affinityfor the folate receptors [22–24]. Theunique integration of drug targetingand visualization has high potential to address the current challenges incancer therapy. Thus, it is attractive to consider the possibility ofinvestigating a system that combines the biodegradable material,chitosan and GO.

Given that GO can be dispersed as individual sheets in water, it ispossible to obtain a molecular-level dispersion of GO if water is usedas the common solvent for both GO and the polymer matrix.Additionally, it is reported that epoxy groups in GO react withamine group of chitosan by addition, which is apparently an approachto modify GO via attachment with amine. Thus, a novel formulation ofchitosan and GO can be obtained via H-bonding.

1306 D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

Considering the intrinsic properties of chitosan [25–30], it offersthe following advantages: (i) amino (\NH2) groups in chitosanfacilitates cross-linking and ligand attachment for targeted drugdelivery, (ii) the chelation and cation properties of chitosan does notrequire chemical binding with nanoparticles because many surfacesare negatively charged and chitosan is positively charged, promotingelectrostatic interaction, (iii) cationic chitosan-based vesicles caneffectively attach to negatively charged phospholipid bilayer ofcellular membrane, (iv) the presence of lysosomes in cellularendocytosis helps degrade CHI to release the encapsulated drug athigher efficiency upon intracellular uptake of nanoparticles, (v) thesolubility of chitosan in mild acid (endosomal pH 5.3) and insolubilityin physiological pH (7.4) prevents untimely release of encapsulateddrug before the target site is reached, and (vi) its mucoadhesive andbacteriostatic properties prolong the retention to targeted substrates,and discourage bacterial uptake in the gastrointestinal tract. Theobjection here is to extend this background knowledge to tumortargeted drug delivery.

Considering the above outlined advantages of GO and chitosan, wedescribe here the synthesis of a novel folate decorated chitosan-graphene oxide nanocarrier for targeted delivery of anticancer drug,DOX. The synthesis of nanocarrier involved the loading of DOX to GOsurface via π–π stacking interaction, followed by encapsulation of GOwith folic acid conjugated chitosan. This π–π stacking interaction isascribed as a non-covalent type of functionalization that allowscontrolled release of drug [31]. This novel approach is expected tohave a significant impact on nanopharmaceutical product develop-ment, including cancer therapy, as distribution of drug-loadednanocarrier can be visualized in vivo because folate receptors areover-expressed on cancer cells [32].

In our research, we focused on a new family of folate decoratedand GO mediated drug delivery system and describe the chemicalsynthesis of the nanocarrier. Since FTIR is considered as anappropriate tool to confirm the interaction between constituents;chemical synthesis is confirmed through detailed investigation byFTIR. The potential viability of the system is proven by investigatingdrug delivery response in a simulated environment.

Our future work will report on in vivo visualization of drug-loadednanocarrier.

2. Experimental procedure

2.1. Materials

Low molecular weight chitosan (~50 kDa) with 75–85% degree ofdeacetylation (DD), ethanol (≥99.5%), anhydrous dimethyl sulfoxide(DMSO), N,N′-dicyclohexyl carbodiimide (DCC), folic acid (FA), NaOH,acetic acid and dialysis membranes (MW=12,400) were obtainedfrom Sigma-Aldrich, USA. Doxorubicin hydrochloride (DOX) wasobtained from Tecoland Corporation, USA. Graphene oxide waspurchased and used as such.

2.2. Dispersion of graphene oxide

Graphene oxide, synthesized from natural graphite, can be easilydispersed in water. Based on this, a desired amount of graphene oxidepowder (10 mg) was dispersed into 30 mL of distilled water, and wasultrasonically dispersed for 1 h to make a homogenous dispersion ofGO in water.

2.3. Synthesis of biofunctionalized chitosan

Since a high molecular weight CHI with high degree of deacetyla-tion can alter the dispersion ability of graphitic materials [33], lowmolecular weight CHI (~ 50 kDa, DD 75–85%) was used. Since folatereceptors are over-expressed on cancer cells, we have intended to

encapsulate graphene oxide (GO) with folate conjugated CHI. [34].This involved the synthesis of biofunctionalized CHI (folate–chitosanconjugate), followed by the encapsulation of drug (DOX) loaded GOwith biofunctionalized CHI to obtain DOX-GO–chitosan-folate nano-carrier. Chitosan has one amino group and two hydroxyl groups,which facilitate strong electrostatic interaction with negative chargedbiomolecules and GO, promoting uniform and homogenous disper-sion [35,36]. FA is covalently linked with CHI to keep a positive chargeratio of more than +2. Since, FA is difficult to conjugate withpolymers because of the low reactivity of the carboxylic acid group[37], we first activated the carboxyl group of FA.

For this, a solution of DCC and FA in anhydrousDMSOwas preparedand stirred at room temperature to completely dissolve FA (1 h).Subsequently, this solution was added to a solution of 1% (w/v) CHI inacetate buffer (pH 4.7). The resulting solutionwas vigorously stirred atroom temperature in the dark (to prevent photo-degradation) for15 h. Itwas thenbrought to pHof 9 by aqueousNaOHanddialyzedfirstagainst phosphate buffer solution (pH 7.4) for 3 days and then againstwater for 3 days. Finally, the chitosan–folate conjugate was freeze-dried and conjugation was confirmed by studying the characteristicabsorption bands associated with functional groups of FA and CHI,using Fourier Transform Infrared (FTIR) Spectroscopy. The relevantchemical reaction and scheme are presented in Fig. 1.

2.4. Loading of graphene oxide (GO) with doxorubicin (DOX)

DOX (30 mg) and GO (10 mg) were added to 30 mL PBS bufferedsolution (pH7.4) and stirred for 16 h at room temperature in darkness.The product (DOX-GO) was collected by repeated centrifugation andwashing with PBS until the supernatant became color free. Theresulted DOX-GO was freeze-dried. The amount of unbound DOX wasdetermined by measuring the absorbance at 490 nm (the character-istic absorbance of DOX) relative to a calibration curve recorded underidentical conditions, allowing the drug loading efficiency to beestimated.

To quantify free DOX, the centrifuged solution was collected anddiluted to 100 mL with deionized water in a flask. The free DOXweight (Wfree DOX) in the solution was determined by fluorometricmeasurement (Varian Cary Eclipse fluorescence pectrophotometer,Varian Inc., USA). The amount of free DOX was determined by aUV–VIS spectrometer (Jasco V-6300 spectrometer) at wavelength of490 nm. Standard DOX water solution (1.0 mg/100 mL) was preparedfor quantitative analysis. The DOX-loading efficiencywas calculated asfollows:

DOX� loading efficiency %ð Þ = 100 WfeedDOX−WfreeDOXð Þ=WfeedDOX

ð1Þ

The DOX-loading efficiency estimated was~96%.

2.5. Synthesis of DOX-loaded GO–chitosan-folate carrier

Encapsulation of DOX-GOwith FA chitosan conjugatewas achievedby adding 10 mg DOX-GO and 20 mg FA conjugated CHI (CHI-FA) in20 mL PBS buffered solution (pH 7.4) under rapid stirring condition atroom temperature in a dark room. After stirring for 16 h, the products(DOX-GO-CHI-FA) were collected by repeated centrifugation andwashed with PBS (~three times) until the supernatant became colorfree, followed by lyophilization. The DOX-loading efficiency wasdetermined as describe above and was estimated to be~97%. Theschematic illustration of the fabrication of nanocarrier is presented inFig. 1.

DOX

DOX

DOX

a. Preparation of Folic Acid Conjugated Chitosan

b. Encapsulation of Graphene oxide-Doxorubicin Nanohybrid with Folic AcidConjugated Chitosan

c.

Fig. 1. Scheme for the synthesis of nanocarrier (a) conjugation of chitosan with folic acid, (b) loading of graphene oxide with doxorubicin, and (c)encapsulation of graphene oxideloaded doxorubicin with folic acid conjugated chitosan.

1307D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

2.6. In-vitro drug release response

The drug release profile from the synthesized nano-carriers DOX-GO and DOX-GO-CHI-FA was studied at the physiological temperatureof 37 °C and pH of 5.3 (the endosomal pH of cancer cells) and pH of 7.4(the physiological pH). Briefly, 3 mg of the drug carrier was sealed in adialysis membrane tube. The dialyses tube was immersed in 10 mL ofNa2HPO4-KH2PO4 buffer solution with pH of 5.3 or 7.4 and placed in atest tube, followed by placing it in a water bath maintained at 37 °C.An aliquot containing the drug release medium (~2.5 mL) waswithdrawn every 1 h until 12 h and thereafter every 12 h until 72 h.The amount of DOX (Wfree DOX) in the buffer solution was quantifiedusing UV–VIS absorption spectra as described in Section 2.4. Aftereach measurement, the aliquot was poured back to the releasesystem. Given that the measurement time was very short, while thedrug release predetermined time interval was significantly large,the influence of the returned medium on drug release during themeasurement time is expected to be insignificant. All the drug releaseexperiments were repeated at least three times.

The release of drug molecules from the carrier could affect themorphology of the nanohybrids drug carrier, so in order to evaluate

any morphological changes we studied the samples via scanningelectron microscopy (SEM). The DOX-GO–CHI-FA nanohybrid drugcarrier samples before and after drug release (pH 5.3, 37 °C) wereplaced on a stub and sputter coated with gold and examined at 15 keVin a Hitachi S-3000 scanning electron microscope.

2.7. Chemical characterization

The chitosan–folic acid conjugate and the nanocarrier wascharacterized using Fourier transform infrared spectroscopy (FTIR)(FT/IR-480) of FA, CHI, chitosan–folic acid, GO, DOX, DOX-GO andDOX-GO–CHI-FA carriers using a KBr compressed pellet method in thereflection mode at 4 cm−1 resolution. Considering that the delivery ofdrug primarily depends on the nanocarrier, we focused on thecharacterization of products at different steps involved in thefabrication of the nanocarrier by FTIR and UV–VIS spectroscopy. Theinteraction between GO and DOX was further investigated byfluorometric measurement (Varian Cary Eclipse fluorescence spec-trophotometer, Varian Inc., USA). GO, DOX, and GO-DOX in the watersolution (0.1 mg/mL) was determined by fluorescence emission using480 nm as the excitation wavelength.

1308 D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

3. Results and discussion

3.1. Biofunctionalization of CHI with FA: chitosan–folic acid conjugate

Folic acid–chitosan conjugate was synthesized by covalently linkingfolic acid to chitosan via EDC as a carboxyl activating agent. Theconjugation reaction occurs at the amino groups of CHI and carboxylgroups of FA (Fig. 1). The FTIR spectrum for FA, chitosan, and chitosanfolic acid conjugate is presented in Fig. 2. Characteristic FTIR absorptionpeaks of FAwere observed at 3543, 3416, 3324, 2959, 2924, 2844, 1694,1640, 1605, 1484 and 1411 cm−1. The peaks appearing between 3600and 3000 cm−1 are due to the hydroxyl (\OH) stretching and N\Hstretching vibrations bands. The C_O stretching vibration of carboxylgroup appears at 1694 cm−1, while the peak at 1640 cm−1 assigned tothe C_O bond stretching vibration of \CO\NH group. The peakappearing at 1605 cm−1 is due to the bending mode of N\H vibrationand at 1411 cm−1 corresponds to the\OH deformation band of phenylskeleton. The characteristic absorption peak of phenyl ring of FAappeared at 1484 cm−1 [38,39].

The FTIR spectrum of CHI (Fig. 2b) revealed the existence ofstretching vibration of N–H (3440 cm−1), and C\H stretch vibration(2924 cm−1 and 2846 cm−1) functional groups. Peaks at ~1647 cm−1,1540 cm−1 and 1317 cm−1 are characteristic of amide I, II and III,respectively. The representative peaks due to \CH3 symmetricaldeformation appears at 1420 cm−1 and 1383 cm−1, while peaks at1153 cm−1 and 1088 cm−1 are indicative of C\O stretching vibrationsfrom (C\O\C). The saccharide structure of CHI is confirmed by a smallpeak at ~900 cm−1 due to wagging [40].

Successful conjugation of FA to CHI is confirmed by interpreting theFTIR spectrumof chitosan–folic acid conjugate (Fig. 2c), which shows thecharacteristic absorption bands of both CHI and FA. The peak due to thedeformation vibration N\H amide-II of the amine group (1597 cm−1)shifted to a higher frequency at 1605 cm−1 in FA conjugated CHI,confirming the existence of chemical interactions between FA and CHI.

14111484

1605

1640

1694

29593324

2846

1088

1263

1317 900

11531420

1597

1647

2924

3416

3543

3440

a. Folic acid

b. Chitosan

c. Chitosan-folic acid conjugate

2924

1383

2844

1605

1408

3330

2852 1072

12451312

893

1575

1628

1691

293034351380

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Tra

nsm

itta

nce

(ar

b. u

nit

s)

Fig. 2. Fourier transform infrared (FTIR) spectra of (a) folic acid, (b) chitosan, and(c) chitosan–folic acid conjugate.

Moreover, the peaks due to amide-III (1317 cm−1) and C\O stretchingvibrations (1088 cm−1) of CHI were shifted to 1312 and 1072 cm−1,respectively, suggesting thepossibility of a strong interactionbetween FAandCHI. Thecharacteristic FTIRpeaksderived fromFig. 2 are summarizedin Table 1.

3.2. Conjugation of GO with doxorubicin (DOX): DOX-GO

Doxorubicin (DOX) is an anticancer drug and is commonly used totreat tumors [41]. GO has a two-dimensional nano-structure consist-ing of sp2-hybridized carbon containing carboxyl, hybroxyl, andepoxide functional groups [42]. The sp2 hybridized π conjugatedstructure of graphene sheet can form π–π stacking interaction withquinine portion of DOX [43], while amino (\NH2) and hydroxyl(\OH) groups of DOX can also form a strong hydrogen-bondinginteraction with \OH and carboxyl (\COOH) groups in GO.

The FTIR spectra of pristine DOX, DOX-loaded GO (DOX-GO) andDOX-loaded GO encapsulated with CHI-FA conjugate (DOX-GO–CHI-FA)are shown in Fig. 3. The FTIR absorption bands derived from Fig. 3 aregiven in Table 2.

Fig. 3a represents the FTIR spectrum of DOX, in which thecharacteristics quinone and ketone carbonyl peaks of DOX appearedat3334, 2925, 1732, 1620, 1414and1071 cm−1. Thepeak at1545 cm−1

is due to the stretching bands of the N\H group, while the peak at816 cm−1 is due to the stretching bands of C\O\CH3. The peaks at 871and 764 cm−1 are due to the primary amine (NH2) wagging and N\Hdeformation bonds, respectively.

The FTIR spectra of GO are given in Fig. 3b. The peaks at 1730 and1630 cm−1 correspond to the stretching vibration of carboxyl (C_O)and deformation of the hydroxyl (\OH) groups in water present inGO, respectively. The peak centered at 1388 cm−1 is attributed to thedeformation vibration of C\OH bond, while an intense band at1051 cm−1 is due to the stretching vibration of C\O bonds in GO [44].

Table 1Assignment of FTIR spectra of folic acid, chitosan, and folic acid conjugated chitosanpresented in Fig. 2.

Samples IR absorptionbands (cm-1)

Descriptiona

(i) Folic acid 3543 ν (O\H)3416, 3324 ν (N\H…H)2959, 2924, 2844 νas (C\H) and νs(C\H) of \CH2

1694 ν(C_O) from \COOH1640 ν(C_O) from \COOH1605 δ (NH2)1484 Phenyl ring1411 O\H deformation of phenyl skeleton

(ii) Chitosan 3440 νs(N\H)2924 νas(C\H)2846 νs(C\H)1647 ν(\C_O\) amide I1597 amine1420,1383 δ(C\H)1317 ν(\CH3) amide III1263 ν(C\O\H)1153, 1088 νas(C\O\C) and νs(C\O\C)900 ω(C\H)

(iii) Folic acidconjugated chitosan

3435 νs(N\H)3330 ν (N\H…H)2930, 2852 νas (C\H) and νs(C\H) of \CH2

1628 ν(\C_O\) amide I1575 ν(N\H) amide II1536 amine1408 O\H deformation of phenyl skeleton1153 νas(C\O\C) and νs(C\O\C)1072 νas(C\O\C) and νs(C\O\C)

a ν = stretching vibration, νs = symmetric stretching vibration; νas = asymmetricstretching vibration; δ= bending vibration; ω = wagging.

3330

762

874

1545

13802854

1726

1730

871

14141620

1732

1071

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Tra

nsm

itta

nce

(ar

b. u

nit

s)

1583 3334

2925 816

764

1630

1618 1115

3450 2932 1632 1580 1085

a. DOX

b. GO

c. DOX-GO

d. DOX-GO-CHI-FA

1388 1095

1285

1115

Fig. 3. Fourier transform infrared (FTIR) spectra of (a) DOX, (b) GO, (c) DOX-loaded GO,and (d) DOX-loaded GO encapsulated with chitosan–folic acid conjugate.

1309D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

Fig. 3c is the FTIR spectra of DOX-loaded GO. Compared to Fig. 2b,some additional absorption bands are observed at ~1115 and 816 cm−1

(corresponding to the stretching bands of C\O\CH3 fromDOX), and at~874 and 762 cm−1 (corresponding to the primary amine NH2wag andN\H deformation bonds from DOX, respectively). These observationsconfirm the loading of DOX to GO. The obtained results are in closeagreement with an earlier report [45].

Table 2Assignment of FTIR spectra of DOX, GO, DOX loaded GO, and DOX-loaded folic acidconjugated-chitosan encapsulated GO presented in Fig. 3.

Samples IR absorption bands(cm−1)

Descriptiona

(i) DOX 3334, ~1071 Quinone and ketonecarbonyls

1530 ν (N\H) amide I871 ω(N\H)816 ν(C\O\CH3)764 δ (N\H)

(ii) Graphene oxide (GO) 1730 ν(C_O\)1630 O\H deformation

(iii) DOX-loaded GO 1115, 817 νs(C\O\CH3) of DOX874 ω (–NH2) (primary amine)

of DOX762 ν (N\H) of DOX

(iv) DOX-loaded folic acidconjugated-chitosanencapsulated GO

3450 νs(N\H)3330 ν (N\H…H)2932 νas (C\H)2854 νs(C\H)1632 ν (\C_O\) amide I1580 ν(N\H) amide II1408 O\H deformation of

phenyl skeleton1380 δ (C\H) of chitosan1285 ν(C\O\H) of chitosan1085 ν(C\O\C) of chitosan

a ν = stretching vibration, νs = symmetric stretching vibration; νas = asymmetricstretching vibration; δ = bending vibration; ω = wagging.

The FTIR spectrum of DOX-loaded GO encapsulated with chitosan-folic acid conjugate is given in Fig. 3d, in which the stretchingvibration band of C\O\C (~1085 cm−1), stretching vibration band ofC\O\H (~1285 cm−1), and bending vibration band of CH\(~1380 cm−1) appeared and subsequently confirmed the conjuga-tion. The broad peak between 3600 and 3000 cm−1 representshydroxyl (OH) stretching and NH\ stretching vibrations bands.

The above mentioned hypothesis is supported by UV–VIS spectra,in which GO shows its characteristic single broad peak at 230 nm, andwhile DOX solution strongly absorbs at 233, 253, 291, and 480 nm, asshown in Fig. 4. The UV–VIS spectrum of the GO-DOX nano-hybridsolution not only confirms the stacking of DOX onto GO, but alsoshows the change in the absorbance (red-shift) due to the interaction.For example, the peaks of DOX at 233 and 480 nm shifted to 235 and486 nm after hybridized with GO, which are generally believed due tothe ground-state electron donor–acceptor interaction between thetwo components, [46,47], namely GO and DOX. The increase inabsorbance led to the change in the color of the solution from darkbrown to red (Fig. 4 INSET).

In the fluorescence spectra (Fig. 5) free DOX exhibits a fluores-cence emission maximum at ~593 nm. However, GO-DOX exhibitsignificant quenching of ~593 nm emission band at the sameexcitation wavelength. These results indicate that there is a strongπ–π stacking interaction between GO-DOX.

3.3. Drug release response

The loading capacity of DOX was found to be around 1 mg/mg ofGO nano-sheets. Such a high value of drug loading is far beyond thecommon drug carrier materials, such as carbon nano-horns [48] andpolymer vesicles, [49] which are generally below 1 mg/mg atsaturated carrying concentration. The above mentioned observationsindicate that GO is indeed a promising candidate for drug carrierherein, the DOX release from GO-DOX and GO-DOX–chitosan-folateconjugate at temperature of 37 °C in the phosphate buffer solution(pH=5.3 and 7.4) is given in Fig. 6. The experimental parameters liketemperature of 37 °C and pH (7.4 and 5.3) were selected because ofthe resemblance with physiological conditions.

The fate of a drug from a drug carrier depends on variousexperimental factors such as pH, degradation rate, particle size,interaction between drug and the surface and behavior of polymer insolvent [50,51]. In current study,we have chosenDOX as amodel drug todemonstrate the loading and release froma graphene oxide and chitosan

DOX-GO

GODOX

480

486

233

230

235

GO DOX-GO

DOX-GO-CHI-FA

200 300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ab

sorb

ance

Wavelength (nm)

Fig. 4. UV–VIS absorbance spectra of DOX, GO, DOX-GO, and DOX-loaded GOencapsulated with chitosan–folic acid conjugate. The loading of DOX was evidencedby a strong absorption peak centered at 480 nm. The reddish color of the GO-DOX isseen in the solution (INSET).

500 550 600 650 700 7500

2

4

6

8

Inte

nsi

ty (

a.u

)

Wavelength (nm)

GO-DOX

DOX

GO

Excitation wavelength = 480 nm

Fig. 5. Fluorescence spectra of aqueous solution of GO, GO-DOX, and DOX at 480excitation wavelength. The concentration of GO, GO-DOX, and DOX were kept constantas per the loading of DXR on GO.

1310 D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

based nanocarrier.We found that at pH7.4, DOX released at a slower andcontrolled manner from DOX-GO and DOX-GO–CHI-FA system, to theextent of ~7%and11% respectively, after72 h. Theamountof drug releasefrom the chitosan–folic acid encapsulated carrier is lower by ~4% thannon-encapsulated DOX-GO carrier.

It is ascribed to the hydrogen-bonding interaction between DOXand GO, which is more prominent in the neutral conditions, resultingin a controlled release. On the other hand, the release pattern underacidic media indicates that the amount of DOX released in the first24 h is much higher than at neutral conditions. It was found thataround 35% of the total bound DOX was released from the nano-hybrid after 24 h at pH 5.3. Under acidic conditions, the amine(\NH2) groups of DOX get protonated resulting in the partialdissociation of hydrogen-bonding interaction, hence the amount ofreleased DOX from GO is much higher. The interaction between theGO sheet and DOX is due to π–π stacking as the loading of DOX on GOis still high. The obtained results are in close agreement with earlierreport with daunomycin and single-walled carbon nanotubes, [52]this efficient loading and release of DOX indicates strong π–π stacking

0 10 20 30 40

Time (h)50 60 70 80 90

0

10

20

30

40

50

60

70

% C

um

ula

tive

DO

X R

elea

se

DOX-GO-CHI-FA-7.4 DOX-GO-CHI-FA-5.3 DOX-GO-5.3 DOX-GO-7.4

Fig. 6. Cumulative doxorubicin release (%) from the GO and DOX-loaded folic acid-conjugated-chitosan encapsulated GO (DOX-GO–CHI-FA) drug carrier at 37 °C inphosphate buffer solution (pH=5.3 and 7.4). The data points are average of at leastthree experiments. Error bars represent the range over which the values were observed.

interaction between GO and DOX. The loading of DOX onto GO can beattributed to simple π–π stacking, similar to that with carbonnanotubes, but as compared to single-walled carbon nanotubes fordrug loading via π-stacking, GO is advantageous in terms of its lowcost and ready scalability [53].

Following the experiment, furtherworkwas carried out on chitosan-folic acid conjugate at pH 5.3 and it was found that a lower amount ofdrugwas released,which is due to the chitosan–folic acid conjugate thatwas used to encapsulate GO-DOX. This observation offers twopossibilities that can explain the decrease in drug release in thepresence of CHI. First, there is a strong intermolecular force betweenDOX and carrier, while secondly the outer shell of biopolymeric CHIoffers an additional path the drug has to travel across the macromo-lecular chains.

The effect of the encapsulation with CHI-FA conjugate on the drugrelease of DOX from DOX-GO and DOX-GO–CHI-FA drug carriers isshown in Fig. 7. As can be seen, during the first 6 h, a significantamount of drug has been released from the DOX-GO nanocarrier atboth the pH, while encapsulating with CHI-FA conjugate the releaserate of DOX occurred very slowly.

This suggests that the drug is released due to the degradation ofCHI chains besides diffusion through the CHI shell. Furthermore, thechemical interaction between DOX and folic acid through hydrogenbonding could also take place and responsible for slower release ofdrug from chitosan-folic acid conjugate encapsulated DOX-GO (DOX-GO–CHI-FA) nanocarrier. Since, both CHI and DOX are positivelycharged; the possibility of CHI-DOX can be ignored. Moreover, factorslike hydrophobic/hydrophilic interactions and resonance effects thatcan facilitate DOX to form a complex with CHI, stable enough to affectdrug release can be completely ignored [54].

In addition, the release of drug from the encapsulated and non-encapsulated carrier was also probed and it was found that after 72 h,the amount of release was greater at pH of 7.4 was ~18% and 24% fromthe encapsulated and non-encapsulated carrier, respectively. Thishigher release of drug is due to the partial dissociation of hydrogenbonding interaction between DOX and GO as described below.

The loading and release of DOX depends upon the hydrogenbonding interaction with GO and is a function of pH. Under acidicconditions, two kinds of hydrogen bonding can be formed between(a)\COOH of GO and\OH of DOX, and (b) between \OH of GO andthe \OH of DOX. It may be noted that \NH2 of DOX forms \NH3

+

0 10 20 30 40 50 60 70 80 900.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Time (h)

Rat

e o

f D

ox

Rel

ease

, mg

/h

DOX-GO, pH 5.3 DOX-GO, pH 7.4 DOX-GO-CHI-FA, pH 5.3 DOX-GO-CHI-FA, pH 7.4

0 10 20 30 40 50 60 70 80 900.000

0.002

0.004

0.006

0.008

0.010

pH=7.4

pH=5.3

DOX-GO-CHI-FA

Time (h)

Fig. 7. Rate of doxorubicin release (mg h−1) from the GO and DOX-loaded folic acid-conjugated-chitosan encapsulated GO (DOX-GO–CHI-FA) drug carrier at 37 °C inphosphate buffer solution (pH=5.3 and 7.4). The data points are average of at leastthree experiments. Bars represent the range over which the values were observed.

a

b

5 µm

5 µm

Fig. 9. Scanning electron micrographs of DOX-GO–CHI-FA nanohybrid drug carrier afterdrug release at pH 5.3 and 37 °C.

1311D. Depan et al. / Materials Science and Engineering C 31 (2011) 1305–1312

with H+ and cannot participate in hydrogen bonding. Furthermore, atlow pH, the H+ in solution would compete with the hydrogen bond-forming group and weaken the above outlined hydrogen bondinginteraction, which may lead to a greater release of DOX. Also, at therelatively acidic pH of 5.3, degradation of CHI could be greater. AtpH 7.4, the four possibilities of hydrogen bonding include (a)\COOHof GO and \OH of DOX, (b) \COOH of GO and the \NH2 of DOX,(c) \OH of GO and \OH of DOX, and (d) \OH of GO and \NH2 ofDOX. Thus, cumulative hydrogen bonding interaction between GOand DOX is higher at pH of 7.4, consistent with the observationpresented in Fig. 4.

Similarly, the drug release at pH 5.3 is less as comparedwith pH 7.4from the chitosan–folate conjugate–GO-DOX nanocarrier. Thus, therate of drug release can be tuned by varying the chitosan/folic acidratio.

In order to evaluate the morphological changes during the courseof release experiment, the drug carrier was examined by SEM beforeand after drug release. The SEM micrograph suggests the diffusion ofthe drug from the carrier during drug release with a consequentincrease in the size of pores (Fig. 8 and 9). The surface of the carriersample was observed to be nearly non-porous before drug release(Fig. 8), while after drug release the sample shows enough evidence ofsurface segregation and development of significant porosity (5.6±0.4 μm) upon release (Fig. 9), implying the diffusion of the drug fromthe nanohybrids drug-carrier.

In summary, the proposed new family of folate decorated and GOmediated drug delivery system having potential for tumor targetingand controlled release.

4. Conclusions

We have synthesized and demonstrated a novel drug carrier basedon graphene oxide having characteristics of targeted as well ascontrolled release. Graphene oxide was embedded as nanofiller andfacilitate excellent drug loading. The drug release was pH sensitiveand it was found that as compared to pH 7.4, the nanohybrid system

a

b

5 µm

5 µm

Fig. 8. Scanning electron micrographs of DOX-GO–CHI-FA nanohybrid drug carrierbefore drug release.

exhibits higher drug release at pH 5.3, which is ascribed due to thereduced interaction between DOX and drug carrier. This pH-sensitivebehavior along with high drug loading makes this system a promisingcandidate for controlled and targeted drug delivery.

Drug release from chitosan–folic acid encapsulated nanocarrierwas found more promising as compared with its non-encapsulatedcounterpart. It is attributed to the presence of chitosan macromolec-ular chains that undergoes degradation and subsequent diffusion ofdrug to target place and to hydrogenbonding interaction between folicacid and DOX. Moreover, under acidic medium, chitosan degradesfaster, resulting in the disruption of hydrogen bonding. Thus, thisgraphene oxide based nanocarrier system offers practical applicationsin biomedical applications.

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