DSpace at VNU: Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery

DSpace at VNU: Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug D...

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Article Journal of Biomedical Nanotechnology Vol 9, 1416–1431, 2013

www.aspbs.com/jbn

Novel Multifunctional Biocompatible Gelatin-Oleic

Acid Conjugate: Self-Assembled Nanoparticles

for Drug Delivery

Phuong Ha-Lien Tran1 ∗, Thao Truong-Dinh Tran1, Toi Van Vo1,

Chau Le-Ngoc Vo2, and Beom-Jin Lee2 ∗

1International University, Vietnam National University–Ho Chi Minh City, 70000, Vietnam

2College of Pharmacy, Ajou University, Suwon 443-749, Korea

In this work, a novel, biocompatible conjugates of gelatin and oleic acid (GOC) were synthesized by a novel aqueous solvent-based method that overcame challenges of completely contrary solubility between gelatin and oleic acid (OA) The GO nanoparticles (GONs) and Paclitaxel encapsulated nanoparticles (PTX-GON) were prepared by self-assembly

in water These nanoparticles (NPs) were then conjugated with folic acid (FA) for targeting cervical cancer cells (Hela cells) and were characterized for their various physicochemical and pharmaceutical properties Fourier transform infrared spectroscopy (FT-IR) and1H NMR studies indicated the successful synthesis of GOC which showed low critical aggre-gation concentration in water (0.015 mg/ml) All NPs were stable in human blood serum and their mean diameters were below 300 nm suitable for passive targeting Powder X-ray diffraction (PXRD) diffractograms showed the reduction in drug crystallinity and hence, leading to the solubility enhancement of PTX The release of PTX from both PTX-GON and FA conjugated PTX-GON (PTX-FA-GON) was controlled for a long time The cytotoxicity results demonstrated great advantages of PTX-FA-GON and PTX-GON over the conventional dosage form of pacliaxel (Taxol® These results, there-fore, indicate that GOC is a promising material to prepare drug encapsulated NP as a controlled delivery system and PTX-FA-GON is a potential targeted delivery system for cancer therapy

KEYWORDS: Gelatin-OA Conjugate (GOC), Self-Assembled Nanoparticles, Targeted Drug Delivery, Paclitaxel, Folic Acid (FA), Protein Conjugation.

INTRODUCTION

Recent advancements in nanotechnology have had wide

applications in the pharmaceutical industry due to a

num-ber of advantages of placing nano-objects at the desired

position, enhancing sparingly soluble drugs and

control-ling the drug release rate Since nanoparticles (NPs) are the

most widely used nano-objects,1 their generation is well

characterized and has been established by many chemical

scientists.2–5However, their applications in pharmaceutical

formulations are still limited because of their

incompati-bility, low payload and a complicated preparation process

For this reason, biocompatibility and self-assembled nature

are outstanding characteristics for a material to be applied

∗ Authors to whom correspondence should be addressed.

Emails: beomjinlee@gmail.com, thlphuong@hcmiu.edu.vn

Received: 27 October 2012

Accepted: 6 January 2013

in NP production Thus, the authors have performed this research for the investigation of a new self-assembled bio-material which was synthesized by a simple method and can be applied for encapsulating many types of drug with the hope that this material will be widely used in the phar-maceutical industry

An amphiphilic carrier which can form NPs in aqueous medium by self-assembly is more preferable than oth-ers because it has been recognized as a promising nano-system which can be applied to many biotechnological and pharmaceutical ﬁelds with numerous types of drugs.6

These amphiphiles spontaneously form NPs by under-going intra- and/or inter molecular associations between hydrophobic moieties in an aqueous environment The hydrophobic segments make the inner core, which is a host system for various hydrophobic drugs, whereas the hydrophilic segments are oriented toward outer aqueous environments to form the corona or outer shells The shell 1416

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hinders the inactivation of the encapsulated drug by

pre-venting contact with inactivating species in blood.7 These

NPs exhibit unique physicochemical characteristics such

as special rheological features, a narrow size

distribu-tion, considerably lower critical aggregation concentrations

and thermodynamic stability.8 9 However, new

develop-ment of amphiphilic carriers using generally recognized as

safe (GRAS)-listed pharmaceutical excipients are of highly

motivated issue because of safety and diverse applications

in drug delivery and clinical therapy

In this work, the gelatin-OA conjugate (GOC) as a

new biomaterial was originally designed to generate an

amphiphilic structure comprised of two GRAS

materi-als, gelatin and OA, using an activator, monoethanolamine

(MEA) Gelatin was chosen because it is a natural and

biocompatible protein10 and a good wall material for

encapsulation.11It possesses a preferably hydrophilic

prop-erty, which facilitates gelatin solubility in water at body

temperature Moreover, gelatin has multifunctional groups,

including –COOH and –NH2, which promote the

capabil-ity of gelatin to bind with other suitable agents OA is

a biocompatible fatty acid which can not be dissolved in

water and also an agent that induces the stability of many

NP systems.12 13 The aim of our study was to develop

an optimal preparation without the use of an organic

sol-vent for a wide application in manufacturing and for good

health and the environment Because gelatin has amine

groups that cannot bind directly with OA in water, OA

must be activated for a reaction Therefore, MEA was

cho-sen to activate OA for the conjugation of hydrophobic OA

and the hydrophilic parts of gelatin in water as an

inter-mediate substance MEA is a chemical interinter-mediate that is

soluble in water and is used in the manufacturing of

cos-metics and surface-active agents.14Spray drying is selected

herein to collect GOC because it is a common method in

the pharmaceutical industry for rapidly drying solvents to

collect large quantities of samples, and hence, bringing a

further promising application in scalability GOC was then

dispersed in water to obtain self-assembled GO

nanopar-ticles (GON) The surface of the GON was further

conju-gated with folic acid (FA) as a ligand for actively targeting

to cancer cells since receptor-mediated drug targeting to

diseased sites is one of the most promising approaches

in chemotherapy to maximize drug efﬁcacy and minimize

systemic toxicity

The nanoparticles were applied to load paclitaxel (PTX),

an insoluble anticancer agent for controlling drug release

Most of the anticancer drugs have limitations in clinical

administration because the drug delivery of these agents

often requires the use of adjuvants or excipients, which

often cause serious side effects For example, Cremophor

EL (polyethoxylated castor oil) and ethanol are

incor-porating excipients in the pharmaceutical drug

formula-tion of Taxol® for inducing drug solubility but its clinical

usefulness is often hampered by poor water solubility and

potential unwanted side effect.15 The nanoparticles in this research have been expected to offer advantages over con-ventional formulations including the ability of drug protec-tion, targeting the drug to the site of acprotec-tion, and reducing the side effects of chemotherapy The multi-functional nanoparticles, combining tumor targeting and tumor ther-apy might be an ideal alternative carrier for controlled delivery of anticancer drugs, which could not only reduce the harmful side effects of chemotherapeutic agents but also maintain adequate drug levels in the body

MATERIALS AND METHODS

Materials Gelatin was purchased from Kanto Chemical Co., Inc (Tokyo, Japan) OA was purchased from Shinyo Pure Chem-icals Co., Ltd (Osaka, Japan) MEA was from Yakuri Pure Chemicals Co., Ltd Pyrene, TNBS (2,4,6-trinitrobenzene sulfonic acid), FA, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), N -hydroxysuccinimide (NHS),

2-(N -morpholino)ethanesulfonic acid (MES), monoclonal

anti-FA clone VP-52, anti-mouse IgG gold conjugate (20 nm), Cremophor EL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) were purchased from Sigma (St Louis, MO, USA) Hela cells were obtained from the American Type Culture Collection (ATCC, USA) Dulbecco’s modiﬁed Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA and penicillin-streptomycin mixtures were from Gibco BRL (Carlsbad, CA, USA) Paclitaxel (PTX) was obtained from Dae Woong Pharmaceutical Co Ltd., (Seoul, Korea) The solvents were high performance liquid chromatography (HPLC) grade All other chemicals were of analytical grade and were used without further puriﬁcation

Synthesis of NPs

Preparation of GOCs

Gelatin (1 g/100 ml) was dissolved in distilled water at

37C to obtain an aqueous gelatin solution OA was simul-taneously mixed with MEA in distilled water to obtain

a uniform colloidal dispersion at two different concen-trations (g/100 ml) of OA/MEA (1:0.1/0.04; GOC-2:0.3/0.12) The gelatin solution was gradually poured into this OA/MEA colloidal solution while stirring The result-ing clear solution was obtained after 1 h and delivered to the nozzle of the spray dryer at a ﬂow rate of 4 ml/min using a peristaltic pump for spray-drying at 130 C inlet and 80 C outlet temperatures The pressure of the spray air was 3 kg/cm2, and the ﬂow rate of the dry air was approximately 3 mb The diameter of the nozzle was 0.7 mm The powder was washed three times with water and ethanol by centrifugation at 10,000 rpm for 15 min

to remove OA and gelatin remaining The supernatant was discarded, and the powder was dried at room temperature under vacuum

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Preparation of Self-Assembled GONs

The powder of GOCs was dispersed in aqueous media

(water, pH 1.2, pH 6.8 or pH 7.4) under gentle stirring

with a paddle at 50 rpm for different intervals of time

to prepare the self-assembled NPs The resulting GONs

were collected via centrifugation at 40,000 rpm for 20 min

(Beckman Optima™TL Ultracentrifuge, USA) The

super-natant was discarded The pellet was washed three times

with water and lyophilized via freeze-drying at−50C for

2 days (Freeze Dryer, Ilshin Lab Co., Ltd., Korea)

Preparation of Drug-Loaded NPs

GOC-2 was selected in this experiment to prepare

drug-loaded NPs GOC-2 and the drug (PTX) were dispersed

in dichloromethane; the loading amount of drug was 10%

The solution (300 l) was emulsiﬁed in 10 ml of

dis-tilled water and sonicated for 20 min to form an oil/water

emulsion Dichloromethane was evaporated under purge

nitrogen gas for 15 min When present, large

aggre-gates were removed by centrifugation at 1000 rpm for

5 min at 37 C NPs were harvested and washed three

times with distilled water by centrifugation at 40,000 rpm

for 20 min at 37 C The pellets were resuspended in

water, sonicated for 30 s, lyophilized and freeze-dried at

−50C for 2 days The obtained drug-loaded NPs were

PTX-GONs

Preparation of Surface-Functionalized

GON-2 and PTX-GON were selected in this experiment

GONs in MES buffer (1 mg/ml, pH 6.0) were activated by

EDC/NHS for 30 min A solution of FA (pH 7.5) made

by sodium phosphate (0.507 mg/ml) was mixed with the

activated GON for 2 h to obtain FA-GON The FA-GONs

were collected using the same process mentioned above

For PTX-FA-GON, the same process was carried out, but

PTX-GON was used instead

Characterization of GOCs

1H NMR Spectroscopy

The synthesized conjugates were identiﬁed using 1H

nuclear magnetic resonance (1H NMR) The experiments

were performed on a NMR spectrometer type Bruker

Avance 600 MHz, and perdeutero DMSO-d6 was used as

a solvent

Fourier Transform Infrared Spectroscopy FTIR

The spectra of the samples (gelatin, OA, conjugates,

PTX-GON, and PTX-FA-GON) were recorded using an IR

spectrophotometer (Excaliber Series UMA-500, Bio-Rad,

USA) KBr pellets were prepared by gently mixing 1 mg

of the sample with 200 mg KBr Fourier transform infrared

spectra (400–4000 cm−1 were obtained with a resolution

of 2 cm−1

Determination of the Degree of Substitution

The number of amino groups of gelatin that reacted with

OA was determined using the TNBS method Samples (2 mg) were dissolved in 5 ml of reaction buffer, 0.1 M NaHCO3, and 2.5 ml of 0.01% TNBS (2,4,6-trinitrobenzene sulfonic acid) was added and mixed well The samples were incubated at 37 C for 2 h Finally, 1.25 ml of 1 N HCl was added to each sample and mixed, and the absorbance of the solutions was measured at

335 nm The free amino groups were determined for pure gelatin, GOC-1 and GOC-2 The degree of substitution (DS) was calculated as follows: DS= A G − A N /A G×

100, whereA G andA N are the absorbance of gelatin and the NPs, respectively, at 335 nm DS was deﬁned as a per-centage of the number of reacted amino groups relative to the number of free amino groups in pure gelatin

To specify how many pairs of GOCs are in one NP, the amount of particles contained in 1 mg of NPs was estimated based on the particle size and density of the polymer Based on the results of the above TNBS method with the amount of reacted amine groups withdrawn from the calibration of cystine, the number of gelatin-OA pairs presented on one particle was determined

Characterization of NPs

Particle Size Measurements and Zeta Potential

The average particle size of self-assembled NPs was mea-sured using a PAR-III Laser Particle Analyzer System (Otsuka Electronics, Japan) All measurements were per-formed in triplicate with a He–Ne laser light source (5 mW) at a 90 angle

The zeta potential of NPs was measured using an Electrophoretic Light Scattering Spectrophotometer 8000 (Otsuka Electronics, Japan) operated at −28.3 V/cm,

−0.1 mA and 28C.

The sample concentration was maintained at 1 mg/ml in distilled water

Morphology of NPs

The solution of self-assembled NPs (1 mg/ml) was placed

on a copper grid to observe the morphology using trans-mission electron microscopy (TEM) (LEO 912AB-100, Carl Zeiss, Korea Basic Science Institute-Chuncheon) The samples of NPs were stained by 2% sodium phospho-tungstate (PTA, pH 7.2) for 40 seconds and dried in a vac-uum dryer at room temperature Thereafter, the grid was examined using a transmission electron microscope For TEM characterization of surface-functionalized NPs with the FA ligand on the surface, further steps for sam-ple preparation were required The presence of the ligand

on the surface of NPs was detected through the pres-ence of a gold NP probe The surface-functionalized NPs were incubated with a mouse anti-FA monoclonal antibody followed by incubation with a 10 nm gold-labeled goat

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anti-mouse IgG The NPs were initially incubated with a

10% BSA solution in PBS for 1 h and then with a

folate-speciﬁc anti-FA antibody for 1 h Unbound antibody was

removed by washing the particles with PBS Particles were

incubated with gold-labeled IgG for 1 h, and unbound gold

particles were removed by washing twice with PBS

High resolution transmission electron microscopy

(HRTEM) (Korea Advanced Institute of Science and

Tech-nology, Daejeon) was also applied to observe the

morphol-ogy of NPs

Measurement of Critical Aggregation

Concentration CAC

The critical aggregation concentration (CAC) of GOC was

determined using a probe ﬂuorescence technique in which

pyrene was used as a hydrophobic probe.9A series of vials

were prepared as follows Pyrene was dissolved in

ace-tone, and the concentration was controlled at 6× 10−7 M.

After evaporation to remove the acetone at 50 C, 5 ml

of different concentrations of the GOC solution (0.00025,

0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25,

0.5 and 1 mg/ml) was added into pyrene Sonication

was performed for 2 h to equilibrate the pyrene and the

NPs The ﬂuorescence spectrum was obtained with a

ﬂu-orescence spectrophotometer (Perkin Elmer Asia LS-55B

M-2721, USA) The excitation wavelength was 336 nm,

and the emission spectra of pyrene were in the range

of 35–450 nm The slit opening for excitation and

emis-sion was set at 10 nm and 5 nm, respectively For the

calculation of CAC, the intensity ratio measurement of

the ﬁrst energy band (374 nm, I1) to the third energy

band (385 nm, I3) in the emission spectra of pyrene was

determined

Powder X-Ray Diffraction PXRD

Powder X-ray diffraction patterns of the samples (Gelatin,

OA, conjugates, PTX-GON, PTX- FA-GON) were

ana-lyzed with a D5005 diffractometer (Bruker, Germany)

using CuK radiation at a voltage of 40 kV and a

cur-rent of 50 mA The powder samples were scanned in steps

of 0.02 from 5 to 60 (diffraction angle 2 with a rate

of one second per step using a zero background sample

holder

Determination of Drug Loading

Content and Encapsulation Efﬁciency

After the supernatant was gathered and the washings were

collected from the NP preparations, the drug loading

con-tent and encapsulation efﬁciency of the NPs were

deter-mined indirectly by HPLC analysis (Waters™, USA) with

a reverse phase column (150× 46 mm, Luna 5u C18

100 A) and a 20 l injection volume For PTX analysis,

the mobile phase consisted of a 55:45 (% v/v) mixture of

acetonitrile and water; the ﬂow rate was 1.0 ml/min, and

the detection wavelength was 227 nm

In Vitro Drug Release

Drug-loaded NPs (1 mg of PTX) were dispersed in 10 ml

at pH 7.4 in screw-capped tubes and placed in an orbital shaker maintained at 37C and shaken at 100 rpm At pre-determined time intervals, 0.5 ml samples were withdrawn for analysis and the same amount of medium was replaced The samples were centrifuged at 40,000 rpm for 20 min and the supernatant was taken for HPLC analysis to deter-mine the drug release The HPLC conditions were per-formed using the same process described in the above section

Biostability Study The stability of GONs with and without the targeting ligand and the NPs loaded with PTX were evaluated by mixing in human blood serum The particle size was con-tinuously monitored by DLS for 24 h Tests in serum were conducted at 37 C to mimic physiological conditions Cancer Cell Killing Effect

Hela (human cervical carcinoma) cells were cultured at

37 C in a humidiﬁed 5% CO2 incubator The cul-ture medium was Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin G sodium and 100 g/ml

streptomycin sulfate A PTX stock solution (6 mg/ml) was prepared by dissolving PTX in ethanol with an equal vol-ume of Cremophor EL, followed by sonication for 30 min, which is the process used for the preparation of Taxol®15

Hela cells were seeded in 96-well plates at a density of

1× 104 cells/well in 200 L of culture media and

incu-bated for 24 h to allow the cells to attach to the dish The growth medium was removed, and the cells were washed twice with PBS to remove the residual growth medium The cells were incubated with 200 L of fresh medium

containing drug-loaded NPs (e.g., GON and PTX-FA-GON), blank NPs or Taxol® at different concentra-tions (concentration of PTX: 2.5, 25, 250, 2500, 12500,

or 25000 ng/mL) for 24 h, 48 h and 72 h at 37 C At the determined time, the cells were washed twice with PBS to eliminate the remaining drug, and 200 L of the

MTT solution (1 mg/ml in PBS) was added to each well Cells were incubated for a further 4 h at 37 C, and the medium was carefully removed Formazan was dissolved

in isopropanol and incubated for 30 min at 37 C The samples were covered with tinfoil and gently shaken on

an orbital shaker for 15 min The absorbance was mea-sured on a VERSAmax tunable microplate reader (USA)

at a wavelength of 570 nm Experiments were measured

in quadruplicate

Cell viability= OD treated/OD control × 100% (OD treated: cells treated with NPs or Taxol®; OD control:

untreated cells)

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Regression analysis for dose-response curves was

per-formed using SigmaPlot version 11.0 (Systat Software

Inc., Oint Richmond, CA, USA) to estimate the IC50 (half

maximal inhibitory concentration)

RESULTS AND DISCUSSION

Synthesis and Identiﬁcation of GO NPs

When gelatin, OA and MEA were introduced into water

for conjugation, the resultant solution exhibited transparent

at the ratio of OA to MEA 1:0.4 or lower However,

with-out gelatin, OA was not completely soluble regardless of

the amount of MEA, so the transparent solution indicated

an interaction between gelatin and OA Therefore, the

formulations of two conjugates (GOC-1 and GOC-2 for

the low and high concentration of OA/MEA, respectively)

were prepared with the aim of investigating the changes

in conjugates or NPs upon increasing amounts of OA

The solution was spray-dried at various inlet temperatures,

such as 80 C, 100 C and 130C The optimal

temper-ature was 130C because the excessive MEA evaporated

completely above 100 C.16 Moreover, this temperature

ensured the complete volatilization of water to produce dry

powder and allow amide bond formation from the

dehy-dration of the salts formed by gelatin’s amine groups and

OA’s carboxylic groups GOC was puriﬁed with ethanol

and water to eliminate free OA and gelatin The puriﬁed

GOC was introduced to water for self-assembly GONs

were collected by a series of steps as follows: repeated

ultracentrifugation, washing with water to eliminate

exces-sive gelatin and ﬁnally, freeze-drying Figure 1

(supple-mentary data) shows the synthesis of the GOC and the

formation of GON Furthermore, Table I shows that the

solubility of GOC was different from that of pure gelatin

(the solubilities of GOC-1 and GOC-2 were quite

simi-lar), which indicates a property change of GOC compared

to pure gelatin As observed after storage for 1 month,

the properties of GOC-1 and GOC-2 were not changed

Herein, the solubility checked within 1 month is mentioned

as the ﬁrst consideration of the system’s stability Other

aspects will be mentioned below

GOCs were characterized using FTIR and 1H NMR

(Fig 2) The peak changes of GOC-1 and GOC-2 showed

a similar pattern The frequency of the C O bonds of

OA and gelatin in FTIR spectra presented at 1709 and

1638 cm−1, respectively GOC-1 and GOC-2 had a new

peak at 1640 cm−1 and 1642 cm−1, respectively, which

was different from the C O peaks of gelatin and OA

in both peak shape and position This result indicated the

formation of an amide between the NH2 group of gelatin

and the C O of OA If a molecule is conjugated, the

strong C O absorption is shifted to the right.17 Herein,

the C O peak of OA at 1709 cm−1 was shifted to the

right by approximately 70 cm−1 It may also be noted that

the peak of GOC-1 and GOC-2 observed at 1640 cm−1and

1642 cm−1, respectively, overlapped with the amide I band

of gelatin, which was evident from an increased inten-sity of this peak An N–H bending vibration of gelatin at

1535 cm−1was also observed for GOC-1 and GOC-2, but the intensity was increased, which implied an overlap An N–H stretching of gelatin at 3303 cm−1 was also observed for GOC Bands at 1338 and 1240 cm−1, which indicated the in-phase combination of C–N stretching and C O bending vibration, also appeared in the GOC-1 and GOC-2 spectra The NMR spectra also veriﬁed the OA binding with gelatin through the presence of OA proton peaks in the GOC spectra, which was a prominently displayed peak

of the alkene bond –CH CH– of OA at 5.32 ppm The disappearance of the proton peak of the –COOH group of

OA at 12 ppm in the GOC spectra indicated the reaction of –COOH groups with gelatin amine groups to form amides The number of amino groups of gelatin reacted with

OA was determined to reveal how many molecules of

OA and gelatin bind each other in GOC-1 or GOC-2 Therefore, the formation of NPs composed of gelatin and

OA can be observed The principle of this calculation is based on the free number of amino groups because their absorbance can be measured by UV-spectroscopy using the TNBS method.18The reacted number of amino groups of GOC can be determined indirectly from the determination

of pure gelatin and the free amino groups remaining in 1 and 2 The substitution percentage of

GOC-1 and GOC-2 were 2752 ± 063% and 6007 ± 101%,

respectively Additionally, gelatin possesses about 33 reac-tive amino groups per gelatin molecule of 1.000 amino acids.19 20 Consequently, the experiment exposed about 9

or 20 amino groups per gelatin molecule of 1.000 amino acids reacting with OA for GOC-1 or GOC-2, respec-tively In other words, 1 gelatin molecule of 1.000 amino acids was binding with 9 OA molecules for GOC-1 and 20

OA molecules for GOC-2 To determine the formation of GOCs into NPs and how many GOC molecules (1 GOC molecule composed of 1 gelatin molecule attached to 9 or

20 OA molecule as mentioned above) could form one NP,

1 mg of NPs was estimated to contain how many parti-cles The conjugates under storage after freeze-drying were used, and the determination was based on the particle size and density of the conjugates.21 The density of GOC-1 and GOC-2 measured in acetone was 0.37 and 1.62 g/cm3, respectively Considering that the average particle sizes of GON-1 and GON-2 were 150 nm and 200 nm, respec-tively, an average of 1 mg of GOC-1 and GOC-2 was estimated to contain 15 × 1012 and 15 × 1011 particles, respectively Based on the known average weight of

GOC-1, GOC-2 and the average weight of one nanoparitcle, the GON-1 and GON-2 was estimated to contain about 11,709 and 77,684 GOCs, respectively

Characterization of GONs Morphology, particle size distribution and zeta potential of GONs under various conditions were evaluated The his-tograms of the particle diameter (Fig 3) revealed that most

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-NH 2 : Gelatin

HOOC-HOOC- : Oleic acid

-NH 2 : Folic acid

C H HC C H HC C N

OH

C H C

H C N

H C H C

N

O

CH 2

CH 2 NH

C NH 2

NH 2

O

H O

CH 2

C O

O

-O

O H

O C O

C O H C H

H C O H C

CH 3 N

gelatin

GO conjugates

Figure 1 Illustration of gelatin-OA conjugates’ synthesis and FA attached NPs.

of the GON-1 and GON-2 dispersed in water for 1 h were

152–182 nm and 196–236 nm in diameter, with an average

value of approximately 170 nm and 220 nm, respectively

Figure 4(a) shows that the particles dispersed in water for

1 h were spherical in shape for both of the NPs formed from GOC-1 and GOC-2 (GON-1 and GON-2, respec-tively), with diameters in the range of 150–200 nm The fact that NP size in DLS was a little larger than the ones

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Table I Solubility property of GOCs in various solvents.

In organic solvent In mixture of water

In water (methanol, acetonitrile, etc.) and organic solvent

Gelatin Insoluble (2 phases: Soluble Insoluble (2 phases: Insoluble (2 phases: Insoluble (2 phases:

gelatin solid/water) gelatin solid/solvent) gelatin solid/solvent) gelatin solid/solvent) GOC-1 Insoluble (turbid Insoluble (turbid Sparingly soluble/Soluble Sparingly soluble Soluble

solution) solution) GOC-2 Insoluble Insoluble Sparingly soluble/Soluble Sparingly soluble Soluble

(turbid solution) (turbid solution)

observed in the TEM images is reasonable because the

TEM images depicted the size in the dried state of the

sam-ple, but the laser light scattering method involved the

mea-surement of size in the hydrated state The increased

amount of OA in the GON-2 induced larger particles

com-pared to GON-1 This result is reasonable because the

hydrophobic section of NPs from OA was increased, which

led to an enlargement of the NPs Because the behaviors

of GON-1 and GON-2 formation and the characterization

in different temperatures and pH conditions were almost

identical, only the images of GON-2 are illustrated GONs

in the self-assembling process with various temperatures

(37 C and 50 C), times (intervals within 1 h and after

storage at room temperature for 1 month) or pH conditions

(water, pH 1.2, pH 6.8 and pH 7.4) exhibited almost

iden-tical TEM images with round-shaped particles Figure 4(b)

wavenumber (cm–1)

1000 2000

3000 4000

1642

1640

1709

1638

gelatin

oleic acid

GOC-1

GOC-2

1535

1240 1338

gelatin

OA

GOC-1

GOC-2

Figure 2 (a) FT-IR spectra of pure gelatin, OA, GOC-1 and GOC-2 (b) NMR spectra of pure gelatin, OA, GOC-1 and GOC-2.

shows that the morphology of GON was not changed within 1 h at 37C in water or after storage of 1 month The pH of the medium had no apparent effect on the mor-phology of NPs (Fig 4(c)) However, pH seemed to affect the size of NPs because lower pH values resulted in NPs with a little smaller size Figure 4(d) shows that a tem-perature of 50 C did not affect the morphology of NPs after 10 min in water Body temperature (37 C) which renders the NPs eligible for any therapeutic treatment was selected to examine the NPs over time Figure 4(e) shows the morphology of GON according to HRTEM, the dif-ferent parts of OA and gelatin are clearly observed This result afﬁrmed the highly expressed core–shell structure of the GONs

Furthermore, Table II shows the particle size distri-bution and zeta potential of GONs using the conjugate

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GON-2

Diameter (nm)

0

5

10

15

20

25

30

GON-1

Diameter (nm)

0

5

10

15

20

25

30

Figure 3 Particle size distribution of GON-1 and GON-2 in

water after 1 h.

GOC-1 and GOC-2 under various conditions DLS

analy-sis also determined that the polydispersity index (PDI) was

not above 0.15 reﬂecting a narrow dispersion22 of GONs

Additionally, GONs had a tendency to slightly decrease in

size at lower pH values This tendency was also observed

in a previous study in which gelatin particle size was

decreased at pH values lower than two.23 However, NP

size was not affected by temperature and had a negligible

variation as a function of time, indicating the stability of

GONs

NP stability was conﬁrmed using a zeta potential

eval-uation of the GONs in aqueous media The zeta potential

of the GONs was negatively charged at values greater than

−30 mV and was not varied by the temperature and time

except pH, indicating a prediction of NP stability because

the surface charge resists the aggregation of the particles.24

This result also indicated that most of the amine groups of

gelatin took part in the reaction with OA, as demonstrated

by the FTIR and1H NMR spectra Consequently, the

sur-face of the GONs primarily stemmed from the COOH

groups of gelatin which is partly hydrolyzed into COO−

and electrostatically stabilized the particle with an anionic

surface charge It is possible that the number of carboxylic

groups present was far greater than the number of remain-ing amine groups present on the surface Therefore, the zeta potential at pH 1.2 exhibited a low positive charge on the surface of NPs, which was attributed to the remain-ing amine groups The zeta potential of the GOs at other

pH values, such as pH 6.8 or 7.4, and at 50 C was not signiﬁcantly different from the zeta potential in water, which indicated the stability of the conjugates Table II also shows the stability of the GONs after 1 month, and the identical particle size distribution and zeta potential were reserved under storage

Stability of GONs was also exposed through criti-cal aggregation concentration (CAC) below which self-assembled systems found in disassemble and kinetic stability Figure 5 shows I1/I3of pyrene emission spectra

as a function of the concentration of GOC-1 and GOC-2, respectively, in water The CAC values of GOC-1 and GOC-2 are 0.023 mg/ml and 0.015 mg/ml, respectively, which is signiﬁcantly lower than the critical micelle con-centration (CMC) of sodium dodecyl sulfate (SDS) in water (2.3 mg/ml)25 and OA in water (0.2033–0.9886 mg/ml).26

The low CAC value, expected to achieve the stable self-assembled NPs at a dilute condition such as body ﬂuid,

is one of the important characteristics for polymeric amphiphiles as a drug delivery carrier.9 18 27 It means that

GOC-1 and GOC-2 can form stable GONs under highly diluted condition Moreover, the lower CAC of GOC-2 was considered herein as a parameter indicating the presence

of the higher amount of OA in the NPs because the CAC values are lowered with increasing hydrophobic moieties.28

Taken together, these results show that the GONs pos-sessed good stability

Characterization of the Functionalized

NP and Drug Loaded NPs Because GON-2 possessed more hydrophobic moiety,

it was chosen in the further processes for attaching the lig-and FA on the surface lig-and loading the model anticancer drug, paclitaxel (PTX) Functionally modiﬁed NPs with

FA (FA-GON) containing PTX (PTX-FA-GON) were also prepared to actively target cancer cells for the speciﬁc recognition of the soluble form of the folate receptor expressed on the surface of cancer cells The effectiveness

of a cancer therapy depends on the ability of the therapeu-tic to eradicate the tumor while affecting as few healthy cells as possible Interestingly, FA, a vitamin whose recep-tor is frequently over-expressed on the surface of human cancer cells and is used as a tumor marker, is highly restricted in most normal tissues.29 30 Therefore, the

devel-opment of delivery systems that can preferentially localize agents to the tumor site has been a recent focus of research Because most of the amine groups of gelatin were used, ligand-targeted NPs with an FA attachment were designed using EDC/NHS, a crosslinking agent soluble in water, to activate the carboxylic groups of gelatin for the reaction of

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(a)

OA gelatin

(b)

(c)

(d) (e)

50 ºC

(f)

45 min

Figure 4 TEM morphology of self-assembled NPs under various conditions (a) GON-1 and GON-2 at 37C in water after 1 h; (b) GON-2 at 37oC in water as a function of time and after storage for 1 month; (c) GON-2 at various pH at 37C after 10 min; (d) GO-2 at 50C after 10 min; (e) HR-TEM of GON-2; (f) TEM image of NPs with FA GON); (g) HRTEM of NPs with FA (FA-GON) in which the arrow points out the black spots on the surface of NPs attributed to gold NPs, indicating the position of FA; (h) HRTEM of gold NPs on the surface of NPs with FA (FA-GON); (i) HRTEM image of NPs without FA containing PTX (PTX-GON).

the amine functional groups of FA As shown in Figure 1,

a superior characteristic of functionalized NPs with a

hydrophilic surface is the capability of transporting the

therapeutics to the target tissues or cells because they can

escape mononuclear phagocytes, macrophages and

retic-uloendothelial systems (RES) in the blood and organs.31

The dense surface concentration of hydrated polymer

chains sterically hinders protein adsorption and

opsoniza-tion to exhibit prolonged circulaopsoniza-tion times in in vivo.32

The attachment of FA is further elucidated through other characterization techniques Figure 4(f) shows a uniformly spherical shape To verify the presence of FA on the sur-face of NPs, GONs were incubated with an FA anti-body and with a gold-labeled secondary antianti-body (20 nm) 1424

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Table II Physicochemical properties of self-assembled GONs under various conditions.

Particle Polydispersity Zeta

pH Temperature (C) Dispersion time (min) size (nm) index (PDI) potential (mV)

GON-2 175 69 ± 497 0 03 ± 001 4 16 ± 153

GON-2 235 04 ± 507 0 06 ± 002 −3745 ± 055

GON-2 220 54 ± 350 0 08 ± 005 −3819 ± 193

GON-2 205 72 ± 330 0 11 ± 004 −3048 ± 023

10 GON-1 158 50 ± 442 0 10 ± 004 −3878 ± 151

GON-2 200 06 ± 599 0 09 ± 002 −4025 ± 098

15 GON-1 156 07 ± 668 0 08 ± 002 −3659 ± 111

GON-2 215 45 ± 570 0 02 ± 002 −3545 ± 212

30 GON-1 160 37 ± 554 0 06 ± 003 −4307 ± 518

GON-2 218 22 ± 300 0 09 ± 000 −3889 ± 056

45 GON-1 164 53 ± 518 0 03 ± 004 40 82 ± 019

GON-2 213 66 ± 479 0 05 ± 004 −3756 ± 080

60 GON-1 169 56 ± 543 0 09 ± 002 −4485 ± 566

GON-2 219 65 ± 220 0 05 ± 005 −4046 ± 279

GON-2 220 00 ± 599 0 04 ± 002 −3470 ± 048 Storage at room temperature/1 month,

then dispersing at 37 C/10 min/water GON -1 168 44 ± 274 0 07 ± 002 −3566 ± 095

GON -2 220 25 ± 416 0 09 ± 001 −3229 ± 074

to recognize the position where FA attached to the surface

of the NPs FA bound with the gold probe is shown in

Figure 4(g), in which the black spots (gold NPs) show the

place where FA attached to the surface of the NPs,

con-ﬁrming the presence of this ligand on the surface of GONs

HRTEM, which provided a closer view of FA-GONs on

the surface, showed the crystal lattice of the gold NPs

(Fig 4(h)) The FA-GON had a mean diameter of

approx-imately 200 nm

PTX is one of the most effective anticancer agents

However, this drug has high incidences of adverse

reac-tions, including neurotoxicity, myelosuppression and

aller-gic reactions.8Cremophor EL (polyethoxylated castor oil),

the excipient incorporated for increasing drug

solubil-ity, is also a cause to hamper the clinical usefulness of

PTX.15 Additionally, PTX is a hydrophobic drug with

a poor aqueous solubility of approximately 1 g/ml.33

For this reason, the controlled release behavior of PTX

from polymeric NPs to suppress these adverse reactions

has been highly recommended One common method to

prepare drug-encapsulated NPs is the solvent

evapora-tion method in which the polymers containing the drug

are solubilized in one of the phases of an emulsion, and

the polymer is precipitated to entrap the drug out of

the solvent during solvent evaporation, which leaves the

drug-loaded particles suspended in the residual solvent.34

Generally, a surfactant must be used to make

small-sized NPs in this emulsion solvent evaporation system,

but it can absorb to the NP surface to signiﬁcantly

affect particle size, the biodegradation rate, the biodis-tribution and physicochemical properties.35 36 Therefore,

a surfactant-free nanoparticulate system has been sug-gested for preparation The GOCs are fortunately com-posed of components that possess surfactant properties for stabilization, which leads to spontaneously stabilized NPs without the necessity for surfactant Therefore, the encap-sulation of PTX in the GONs is performed in appropriate conditions with high efﬁciency (Table III) The entrap-ment of the drug involves the incorporation of the drug into the hydrophobic cores of GONs due to hydropho-bic interactions, as previously shown in other research.37 38

Moreover, the solubility of PTX in the current drug-loaded nano systems was increased compared to the pure drug (Table III)

The in vitro release proﬁle of PTX-loaded NPs

with (PTX-FA-GON) or without FA (PTX-GON) was investigated in PBS (pH 7.4) at 37 C (Fig 6) and com-pared with the pure drug The cumulative percentage release from PTX-FA-GON and PTX-GON was approxi-mately 55% and 45%, respectively, for 6 days and reached 100% for both conjugates after 1 month, which showed the potential of the NPs as a controlled drug delivery system The lower cumulative release of PTX from PTX-FA-GON was perhaps due to the presence of the ligand on the NP surface, which slightly increased the diffusion path On the other hand, drug release from Taxol® has been reported previously to be over 80% for PTX after 4–6 h.39 40 These

results indicated that the GONs in this study could be

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