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...

Article Journal of Biomedical Nanotechnology Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America 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 ∗ International University, Vietnam National University–Ho Chi Minh City, 70000, Vietnam College 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) and H NMR studies indicated the successful synthesis of GOC which showed low critical aggreDelivered by Publishing Technology to: Rice University, Fondren Library gation concentration in water (0.015 mg/ml) All NPs On: wereSat, stable human serum and their mean diameters were IP: 63.142.225.118 10inOct 2015blood 07:05:21 below 300 nm suitable for passive targeting Powder X-ray diffraction (PXRD) diffractograms showed the reduction in Copyright: American Scientific Publishers 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, therefore, 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 number of advantages of placing nano-objects at the desired position, enhancing sparingly soluble drugs and controlling 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.2–5 However, their applications in pharmaceutical formulations are still limited because of their incompatibility, 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: January 2013 1416 J Biomed Nanotechnol 2013, Vol 9, No in NP production Thus, the authors have performed this research for the investigation of a new self-assembled biomaterial 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 pharmaceutical industry An amphiphilic carrier which can form NPs in aqueous medium by self-assembly is more preferable than others because it has been recognized as a promising nanosystem which can be applied to many biotechnological and pharmaceutical fields with numerous types of drugs.6 These amphiphiles spontaneously form NPs by undergoing 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 1550-7033/2013/9/1416/016 doi:10.1166/jbn.2013.1621 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery hinders the inactivation of the encapsulated drug by prepotential unwanted side effect.15 The nanoparticles in this venting contact with inactivating species in blood These research have been expected to offer advantages over conNPs exhibit unique physicochemical characteristics such ventional formulations including the ability of drug protecas special rheological features, a narrow size distribution, targeting the drug to the site of action, and reducing tion, considerably lower critical aggregation concentrations the side effects of chemotherapy The multi-functional and thermodynamic stability.8 However, new developnanoparticles, combining tumor targeting and tumor therment of amphiphilic carriers using generally recognized as apy might be an ideal alternative carrier for controlled safe (GRAS)-listed pharmaceutical excipients are of highly delivery of anticancer drugs, which could not only reduce motivated issue because of safety and diverse applications the harmful side effects of chemotherapeutic agents but in drug delivery and clinical therapy also maintain adequate drug levels in the body In this work, the gelatin-OA conjugate (GOC) as a new biomaterial was originally designed to generate an MATERIALS AND METHODS amphiphilic structure comprised of two GRAS materiMaterials als, gelatin and OA, using an activator, monoethanolamine Gelatin was purchased from Kanto Chemical Co., Inc (MEA) Gelatin was chosen because it is a natural and (Tokyo, Japan) OA was purchased from Shinyo Pure Chem10 biocompatible protein and a good wall material for icals Co., Ltd (Osaka, Japan) MEA was from Yakuri Pure 11 encapsulation It possesses a preferably hydrophilic propChemicals Co., Ltd Pyrene, TNBS (2,4,6-trinitrobenzene erty, which facilitates gelatin solubility in water at body sulfonic acid), FA, 1-ethyl-3-(3-dimethylamino-propyl) temperature Moreover, gelatin has multifunctional groups, carbodiimide (EDC), N -hydroxysuccinimide (NHS), including –COOH and –NH2 , which promote the capabil2-(N -morpholino)ethanesulfonic acid (MES), monoclonal ity of gelatin to bind with other suitable agents OA is anti-FA clone VP-52, anti-mouse IgG gold conjugate a biocompatible fatty acid which can not be dissolved in (20 nm), Cremophor EL and 3-(4,5-dimethylthiazol-2-yl)water and also an agent that induces the stability of many 2,5-diphenyl tetrazolium bromide) (MTT) were purchased 12 13 NP systems The aim of our study was to develop from Sigma (St Louis, MO, USA) Hela cells were an optimal preparation without the use of an organic solobtained from the American Type Culture Collection vent for a wide application in manufacturing and for good (ATCC, USA) Dulbecco’s Delivered by Publishing Technology to: Rice University, Fondren modified Library Eagle’s medium health and the environment Because gelatin has amine IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 (DMEM), fetal bovine serum (FBS), trypsin-EDTA and groups that cannot bind directly with OA in water, OA Copyright: American Scientific Publishers penicillin-streptomycin mixtures were from Gibco BRL must be activated for a reaction Therefore, MEA was cho(Carlsbad, CA, USA) Paclitaxel (PTX) was obtained from sen to activate OA for the conjugation of hydrophobic OA Dae Woong Pharmaceutical Co Ltd., (Seoul, Korea) The and the hydrophilic parts of gelatin in water as an intersolvents were high performance liquid chromatography mediate substance MEA is a chemical intermediate that is (HPLC) grade All other chemicals were of analytical soluble in water and is used in the manufacturing of cosgrade and were used without further purification metics and surface-active agents.14 Spray 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 nanoparticles (GON) The surface of the GON was further conjugated 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 incorporating excipients in the pharmaceutical drug formulation of Taxol® for inducing drug solubility but its clinical usefulness is often hampered by poor water solubility and J Biomed Nanotechnol 9, 1416–1431, 2013 Synthesis of NPs Preparation of GOCs Gelatin (1 g/100 ml) was dissolved in distilled water at 37 C to obtain an aqueous gelatin solution OA was simultaneously mixed with MEA in distilled water to obtain a uniform colloidal dispersion at two different concentrations (g/100 ml) of OA/MEA (GOC-1:0.1/0.04; GOC2:0.3/0.12) The gelatin solution was gradually poured into this OA/MEA colloidal solution while stirring The resulting clear solution was obtained after h and delivered to the nozzle of the spray dryer at a flow rate of 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 kg/cm2 , and the flow rate of the dry air was approximately 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 to remove OA and gelatin remaining The supernatant was discarded, and the powder was dried at room temperature under vacuum 1417 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery 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 (Beckman Optima™ TL Ultracentrifuge, USA) The supernatant was discarded The pellet was washed three times with water and lyophilized via freeze-drying at −50 C for days (Freeze Dryer, Ilshin Lab Co., Ltd., Korea) Tran et al 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 ml of reaction buffer, 0.1 M NaHCO3 , and 2.5 ml of 0.01% TNBS (2,4,6trinitrobenzene sulfonic acid) was added and mixed well The samples were incubated at 37 C for h Finally, 1.25 ml of 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 = AG − AN /AG × 100, where AG and AN are the absorbance of gelatin and the NPs, respectively, at 335 nm DS was defined as a percentage 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 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 Preparation of Drug-Loaded NPs GOC-2 was selected in this experiment to prepare drugloaded 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 distilled water and sonicated for 20 to form an oil/water emulsion Dichloromethane was evaporated under purge nitrogen gas for 15 When present, large aggregates were removed by centrifugation at 1000 rpm for at 37 C NPs were harvested and washed three times with distilled water by centrifugation at 40,000 rpm for 20 at 37 C The pellets were resuspended in water, sonicated for 30 s, lyophilized and freeze-dried at −50 C for days The obtained drug-loaded NPs were Characterization of NPs PTX-GONs Size Measurements and Zeta Potential Delivered by Publishing Technology to: Particle Rice University, Fondren Library average size of self-assembled NPs was meaIP: 63.142.225.118 On: Sat, The 10 Oct 2015particle 07:05:21 Preparation of Surface-Functionalized Copyright: American Scientific Publishers sured using a PAR-III Laser Particle Analyzer System GONs with FA FA-GONs (Otsuka Electronics, Japan) All measurements were perGON-2 and PTX-GON were selected in this experiment formed in triplicate with a He–Ne laser light source GONs in MES buffer (1 mg/ml, pH 6.0) were activated by (5 mW) at a 90 angle EDC/NHS for 30 A solution of FA (pH 7.5) made The zeta potential of NPs was measured using an by sodium phosphate (0.507 mg/ml) was mixed with the Electrophoretic Light Scattering Spectrophotometer 8000 activated GON for h to obtain FA-GON The FA-GONs (Otsuka Electronics, Japan) operated at −28.3 V/cm, were collected using the same process mentioned above −0.1 mA and 28 C For PTX-FA-GON, the same process was carried out, but The sample concentration was maintained at mg/ml in PTX-GON was used instead distilled water Characterization of GOCs H NMR Spectroscopy The synthesized conjugates were identified using H nuclear magnetic resonance (1 H 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, PTXGON, and PTX-FA-GON) were recorded using an IR spectrophotometer (Excaliber Series UMA-500, Bio-Rad, USA) KBr pellets were prepared by gently mixing mg of the sample with 200 mg KBr Fourier transform infrared spectra (400–4000 cm−1 were obtained with a resolution of cm−1 1418 Morphology of NPs The solution of self-assembled NPs (1 mg/ml) was placed on a copper grid to observe the morphology using transmission electron microscopy (TEM) (LEO 912AB-100, Carl Zeiss, Korea Basic Science Institute-Chuncheon) The samples of NPs were stained by 2% sodium phosphotungstate (PTA, pH 7.2) for 40 seconds and dried in a vacuum 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 sample preparation were required The presence of the ligand on the surface of NPs was detected through the presence 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 J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery anti-mouse IgG The NPs were initially incubated with a 10% BSA solution in PBS for h and then with a folatespecific anti-FA antibody for h Unbound antibody was removed by washing the particles with PBS Particles were incubated with gold-labeled IgG for h, and unbound gold particles were removed by washing twice with PBS High resolution transmission electron microscopy (HRTEM) (Korea Advanced Institute of Science and Technology, Daejeon) was also applied to observe the morphology of NPs 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 37 C and shaken at 100 rpm At predetermined 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 and the supernatant was taken for HPLC analysis to determine the drug release The HPLC conditions were performed using the same process described in the above section Measurement of Critical Aggregation Concentration CAC Biostability Study The critical aggregation concentration (CAC) of GOC was The stability of GONs with and without the targeting determined using a probe fluorescence technique in which ligand and the NPs loaded with PTX were evaluated by pyrene was used as a hydrophobic probe.9 A series of vials mixing in human blood serum The particle size was conwere prepared as follows Pyrene was dissolved in acetinuously monitored by DLS for 24 h Tests in serum were tone, and the concentration was controlled at × 10−7 M conducted at 37 C to mimic physiological conditions After evaporation to remove the acetone at 50 C, ml of different concentrations of the GOC solution (0.00025, Cancer Cell Killing Effect 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, Hela (human cervical carcinoma) cells were cultured at 0.5 and mg/ml) was added into pyrene Sonication 37 C in a humidified 5% CO2 incubator The culwas performed for h to equilibrate the pyrene and the ture medium was Dulbecco’s modified Eagle’s medium NPs The fluorescence spectrum was obtained with a flu(DMEM) supplemented with 10% (v/v) fetal bovine serum orescence spectrophotometer (Perkin Elmer Asia LS-55B (FBS), 100 IU/ml penicillin G sodium and 100 g/ml M-2721, USA) The excitation wavelength was 336 nm, streptomycin sulfate A PTX stock solution (6 mg/ml) was Delivered by Publishing Technology to: Rice University, Fondren Library and the emission spectra of pyrene were in the range prepared dissolving PTX in ethanol with an equal volIP:excitation 63.142.225.118 On: Sat, 10 Octby2015 07:05:21 of 35–450 nm The slit opening for and emisume of Cremophor EL, Copyright: American Scientific Publishers followed by sonication for 30 min, sion was set at 10 nm and nm, respectively For the which is the process used for the preparation of Taxol® 15 calculation of CAC, the intensity ratio measurement of Hela cells were seeded in 96-well plates at a density of the first energy band (374 nm, I1) to the third energy × 104 cells/well in 200 L of culture media and incuband (385 nm, I3) in the emission spectra of pyrene was bated for 24 h to allow the cells to attach to the dish The determined growth medium was removed, and the cells were washed twice with PBS to remove the residual growth medium Powder X-Ray Diffraction PXRD The cells were incubated with 200 L of fresh medium Powder X-ray diffraction patterns of the samples (Gelatin, containing drug-loaded NPs (e.g., PTX-GON and PTXOA, conjugates, PTX-GON, PTX- FA-GON) were anaFA-GON), blank NPs or Taxol® at different concentralyzed with a D5005 diffractometer (Bruker, Germany) tions (concentration of PTX: 2.5, 25, 250, 2500, 12500, using CuK radiation at a voltage of 40 kV and a curor 25000 ng/mL) for 24 h, 48 h and 72 h at 37 C At rent of 50 mA The powder samples were scanned in steps the determined time, the cells were washed twice with of 0.02 from to 60 (diffraction angle with a rate PBS to eliminate the remaining drug, and 200 L of the of one second per step using a zero background sample MTT solution (1 mg/ml in PBS) was added to each well holder Cells were incubated for a further h at 37 C, and the medium was carefully removed Formazan was dissolved Determination of Drug Loading in isopropanol and incubated for 30 at 37 C The Content and Encapsulation Efficiency samples were covered with tinfoil and gently shaken on After the supernatant was gathered and the washings were an orbital shaker for 15 The absorbance was meacollected from the NP preparations, the drug loading consured on a VERSAmax tunable microplate reader (USA) tent and encapsulation efficiency of the NPs were deterat a wavelength of 570 nm Experiments were measured mined indirectly by HPLC analysis (Waters™ , USA) with in quadruplicate a reverse phase column (150 × mm, Luna 5u C18 100 A) and a 20 l injection volume For PTX analysis, Cell viability = OD treated/OD control × 100% the mobile phase consisted of a 55:45 (% v/v) mixture of (OD treated: cells treated with NPs or Taxol® ; OD control: acetonitrile and water; the flow rate was 1.0 ml/min, and untreated cells) the detection wavelength was 227 nm J Biomed Nanotechnol 9, 1416–1431, 2013 1419 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Tran et al Regression analysis for dose-response curves was performed using SigmaPlot version 11.0 (Systat Software Inc., Oint Richmond, CA, USA) to estimate the IC50 (half maximal inhibitory concentration) of gelatin, which was evident from an increased intensity of this peak An N–H bending vibration of gelatin at 1535 cm−1 was 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 RESULTS AND DISCUSSION the in-phase combination of C–N stretching and C O Synthesis and Identification of GO NPs bending vibration, also appeared in the GOC-1 and GOC-2 When gelatin, OA and MEA were introduced into water spectra The NMR spectra also verified the OA binding for conjugation, the resultant solution exhibited transparent with gelatin through the presence of OA proton peaks in at the ratio of OA to MEA 1:0.4 or lower However, withthe GOC spectra, which was a prominently displayed peak out gelatin, OA was not completely soluble regardless of of the alkene bond –CH CH– of OA at 5.32 ppm The the amount of MEA, so the transparent solution indicated disappearance of the proton peak of the –COOH group of an interaction between gelatin and OA Therefore, the OA at 12 ppm in the GOC spectra indicated the reaction of formulations of two conjugates (GOC-1 and GOC-2 for –COOH groups with gelatin amine groups to form amides the low and high concentration of OA/MEA, respectively) The number of amino groups of gelatin reacted with were prepared with the aim of investigating the changes OA was determined to reveal how many molecules of in conjugates or NPs upon increasing amounts of OA OA and gelatin bind each other in GOC-1 or GOC-2 The solution was spray-dried at various inlet temperatures, Therefore, the formation of NPs composed of gelatin and such as 80 C, 100 C and 130 C The optimal temperOA can be observed The principle of this calculation is ature was 130 C because the excessive MEA evaporated based on the free number of amino groups because their completely above 100 C.16 Moreover, this temperature absorbance can be measured by UV-spectroscopy using the ensured the complete volatilization of water to produce dry TNBS method.18 The reacted number of amino groups of powder and allow amide bond formation from the dehyGOC can be determined indirectly from the determination dration of the salts formed by gelatin’s amine groups and of pure gelatin and the free amino groups remaining in OA’s carboxylic groups GOC was purified with ethanol GOC-1 and GOC-2 The substitution percentage of GOCand water to eliminate free OA and gelatin The purified and University, GOC-2 were 27 52 ± 63% and 60 07 ± 01%, Delivered by Publishing Technology to: 1Rice Fondren Library GOC was introduced to water IP: for63.142.225.118 self-assembly GONs On: Sat, respectively 10 Oct 2015Additionally, 07:05:21 gelatin possesses about 33 reacwere collected by a series of steps Copyright: as follows:American repeated Scientific tive amino groups per gelatin molecule of 1.000 amino Publishers ultracentrifugation, washing with water to eliminate excesacids.19 20 Consequently, the experiment exposed about sive gelatin and finally, freeze-drying Figure (suppleor 20 amino groups per gelatin molecule of 1.000 amino mentary data) shows the synthesis of the GOC and the acids reacting with OA for GOC-1 or GOC-2, respecformation of GON Furthermore, Table I shows that the tively In other words, gelatin molecule of 1.000 amino solubility of GOC was different from that of pure gelatin acids was binding with OA molecules for GOC-1 and 20 (the solubilities of GOC-1 and GOC-2 were quite simiOA molecules for GOC-2 To determine the formation of GOCs into NPs and how many GOC molecules (1 GOC lar), which indicates a property change of GOC compared molecule composed of gelatin molecule attached to or to pure gelatin As observed after storage for month, 20 OA molecule as mentioned above) could form one NP, the properties of GOC-1 and GOC-2 were not changed mg of NPs was estimated to contain how many partiHerein, the solubility checked within month is mentioned cles The conjugates under storage after freeze-drying were as the first consideration of the system’s stability Other used, and the determination was based on the particle size aspects will be mentioned below and density of the conjugates.21 The density of GOC-1 GOCs were characterized using FTIR and H NMR and GOC-2 measured in acetone was 0.37 and 1.62 g/cm3 , (Fig 2) The peak changes of GOC-1 and GOC-2 showed respectively Considering that the average particle sizes of a similar pattern The frequency of the C O bonds of GON-1 and GON-2 were 150 nm and 200 nm, respecOA and gelatin in FTIR spectra presented at 1709 and tively, an average of mg of GOC-1 and GOC-2 was 1638 cm−1 , respectively GOC-1 and GOC-2 had a new estimated to contain × 1012 and × 1011 particles, peak at 1640 cm−1 and 1642 cm−1 , respectively, which respectively Based on the known average weight of GOCwas different from the C O peaks of gelatin and OA 1, GOC-2 and the average weight of one nanoparitcle, the in both peak shape and position This result indicated the GON-1 and GON-2 was estimated to contain about 11,709 formation of an amide between the NH2 group of gelatin and 77,684 GOCs, respectively and the C O of OA If a molecule is conjugated, the 17 strong C O absorption is shifted to the right Herein, Characterization of GONs 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 Morphology, particle size distribution and zeta potential of the peak of GOC-1 and GOC-2 observed at 1640 cm−1 and GONs under various conditions were evaluated The histograms of the particle diameter (Fig 3) revealed that most 1642 cm−1 , respectively, overlapped with the amide I band 1420 J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery oleic acid (OA) MEA O C O N H H C CH C O H N O H N H C C H C C CH N H O H N H C C H Heat C N H CH CH CH C C O H N O O- NH MEA H C CH C OH O H N C H C N O C O NH NH gelatin Delivered by Publishing Technology to: Rice University, Fondren Library IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 Copyright: American Scientific Publishers GO conjugates -NH2 : Gelatin HOOCHOOC- -NH2 Figure : Oleic acid : Folic acid Illustration of gelatin-OA conjugates’ synthesis and FA attached NPs of the GON-1 and GON-2 dispersed in water for 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 J Biomed Nanotechnol 9, 1416–1431, 2013 h were spherical in shape for both of the NPs formed from GOC-1 and GOC-2 (GON-1 and GON-2, respectively), with diameters in the range of 150–200 nm The fact that NP size in DLS was a little larger than the ones 1421 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Table I Tran et al Solubility property of GOCs in various solvents In organic solvent (methanol, acetonitrile, etc.) In water In mixture of water and organic solvent Sample Room temperature 37 C Room temperature 37 C Room temperature Gelatin Insoluble (2 phases: gelatin solid/water) Insoluble (turbid solution) Insoluble (turbid solution) Soluble Insoluble (2 phases: gelatin solid/solvent) Sparingly soluble/Soluble Insoluble (2 phases: gelatin solid/solvent) Sparingly soluble Insoluble (2 phases: gelatin solid/solvent) Soluble Sparingly soluble/Soluble Sparingly soluble Soluble GOC-1 GOC-2 Insoluble (turbid solution) Insoluble (turbid solution) observed in the TEM images is reasonable because the shows that the morphology of GON was not changed within h at 37 C in water or after storage of month TEM images depicted the size in the dried state of the samThe pH of the medium had no apparent effect on the morple, but the laser light scattering method involved the meaphology of NPs (Fig 4(c)) However, pH seemed to affect surement of size in the hydrated state The increased the size of NPs because lower pH values resulted in NPs amount of OA in the GON-2 induced larger particles comwith a little smaller size Figure 4(d) shows that a tempared to GON-1 This result is reasonable because the perature of 50 C did not affect the morphology of NPs hydrophobic section of NPs from OA was increased, which after 10 in water Body temperature (37 C) which led to an enlargement of the NPs Because the behaviors renders the NPs eligible for any therapeutic treatment was of GON-1 and GON-2 formation and the characterization selected to examine the NPs over time Figure 4(e) shows in different temperatures and pH conditions were almost the morphology of GON according to HRTEM, the difidentical, only the images of GON-2 are illustrated GONs ferent parts of OA and gelatin are clearly observed This in the self-assembling process with various temperatures result affirmed the highly expressed core–shell structure of (37 C and 50 C), times (intervals within h and after the GONs storage at room temperature for month) or pH conditions Delivered Technology University,Table Fondren Library Furthermore, II shows the particle size distri(water, pH 1.2, pH 6.8 and pH by 7.4)Publishing exhibited almost iden- to: Rice IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 bution and zeta potential of GONs using the conjugate tical TEM images with round-shapedCopyright: particles Figure 4(b) American Scientific Publishers GOC-2 (a) (b) gelatin 1642 GOC-1 OA 1640 oleic acid GOC-1 2839 2925 1709 gelatin GOC-2 13381240 1638 1535 4000 3000 2000 1000 wavenumber (cm–1) Figure 1422 (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 J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery groups present was far greater than the number of remaining amine groups present on the surface Therefore, the zeta potential at pH 1.2 exhibited a low positive charge on 25 the surface of NPs, which was attributed to the remaining amine groups The zeta potential of the GOs at other 20 pH values, such as pH 6.8 or 7.4, and at 50 C was not significantly different from the zeta potential in water, 15 which indicated the stability of the conjugates Table II 10 also shows the stability of the GONs after month, and the identical particle size distribution and zeta potential were reserved under storage Stability of GONs was also exposed through criti0 cal aggregation concentration (CAC) below which self60 80 100 120 140 160 180 200 220 240 260 280 300 assembled systems found in disassemble and kinetic Diameter (nm) stability Figure shows I1 /I3 of pyrene emission spectra as a function of the concentration of GOC-1 and GOC-2, 30 GON-2 respectively, in water The CAC values of GOC-1 and 25 GOC-2 are 0.023 mg/ml and 0.015 mg/ml, respectively, which is significantly lower than the critical micelle con20 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 15 The low CAC value, expected to achieve the stable selfassembled NPs at a dilute condition such as body fluid, 10 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 Delivered by Publishing Technology diluted to: Ricecondition University, Fondren Moreover, theLibrary lower CAC of GOC-2 was 60 80 100 120 140 160 180 200 240 260 280 300 On: Sat, 10 Oct 2015 07:05:21 IP: 220 63.142.225.118 considered herein as a parameter indicating the presence Diameter (nm) Copyright: American Scientific Publishers of the higher amount of OA in the NPs because the CAC values are lowered with increasing hydrophobic moieties.28 Figure Particle size distribution of GON-1 and GON-2 in water after h Taken together, these results show that the GONs possessed good stability GOC-1 and GOC-2 under various conditions DLS analyCharacterization of the Functionalized sis also determined that the polydispersity index (PDI) was 22 NP and Drug Loaded NPs not above 0.15 reflecting a narrow dispersion of GONs Additionally, GONs had a tendency to slightly decrease in Because GON-2 possessed more hydrophobic moiety, size at lower pH values This tendency was also observed it was chosen in the further processes for attaching the ligin a previous study in which gelatin particle size was and FA on the surface and loading the model anticancer decreased at pH values lower than two.23 However, NP drug, paclitaxel (PTX) Functionally modified NPs with size was not affected by temperature and had a negligible FA (FA-GON) containing PTX (PTX-FA-GON) were also variation as a function of time, indicating the stability of prepared to actively target cancer cells for the specific GONs recognition of the soluble form of the folate receptor NP stability was confirmed using a zeta potential evalexpressed on the surface of cancer cells The effectiveness uation of the GONs in aqueous media The zeta potential of a cancer therapy depends on the ability of the therapeuof the GONs was negatively charged at values greater than tic to eradicate the tumor while affecting as few healthy −30 mV and was not varied by the temperature and time cells as possible Interestingly, FA, a vitamin whose recepexcept pH, indicating a prediction of NP stability because tor is frequently over-expressed on the surface of human the surface charge resists the aggregation of the particles.24 cancer cells and is used as a tumor marker, is highly This result also indicated that most of the amine groups of restricted in most normal tissues.29 30 Therefore, the develgelatin took part in the reaction with OA, as demonstrated opment of delivery systems that can preferentially localize by the FTIR and H NMR spectra Consequently, the suragents to the tumor site has been a recent focus of research face of the GONs primarily stemmed from the COOH Because most of the amine groups of gelatin were used, groups of gelatin which is partly hydrolyzed into COO− ligand-targeted NPs with an FA attachment were designed and electrostatically stabilized the particle with an anionic using EDC/NHS, a crosslinking agent soluble in water, to activate the carboxylic groups of gelatin for the reaction of surface charge It is possible that the number of carboxylic GON-1 Distribution (%) Distribution (%) 30 J Biomed Nanotechnol 9, 1416–1431, 2013 1423 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery GON-1 (a) (b) (c) GON-2 15 pH 1.2 Tran et al month 45 pH 6.8 pH 7.4 gelatin Fondren Library (d) Technology to: Rice(e) (f) Delivered by Publishing University, IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 Copyright: American Scientific Publishers 50 ºC OA (g) (h) (i) drug OA gelatin Figure TEM morphology of self-assembled NPs under various conditions (a) GON-1 and GON-2 at 37 C in water after h; (b) GON-2 at 37 o C in water as a function of time and after storage for month; (c) GON-2 at various pH at 37 C after 10 min; (d) GO-2 at 50 C after 10 min; (e) HR-TEM of GON-2; (f) TEM image of NPs with FA (FA-GON); (g) HRTEM of NPs with FA (FAGON) 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 reticuloendothelial systems (RES) in the blood and organs.31 The dense surface concentration of hydrated polymer 1424 chains sterically hinders protein adsorption and opsonization to exhibit prolonged circulation 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 surface of NPs, GONs were incubated with an anti-FA antibody and with a gold-labeled secondary antibody (20 nm) J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Table II Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Physicochemical properties of self-assembled GONs under various conditions pH Temperature ( C) 1.2 37 10 6.8 37 10 7.4 37 10 water 37 10 15 30 45 60 50 Storage at room temperature/1 month, then dispersing at 37 C/10 min/water Particle size (nm) Polydispersity index (PDI) Zeta potential (mV) GON-1 GON-2 GON-1 GON-2 GON-1 GON-2 GON -1 GON-2 GON-1 GON-2 119 60 ± 34 175 69 ± 97 167 43 ± 93 235 04 ± 07 176 42 ± 02 220 54 ± 50 161 45 ± 70 205 72 ± 30 158 50 ± 42 200 06 ± 99 04 ± 01 03 ± 01 08 ± 04 06 ± 02 07 ± 07 08 ± 05 05 ± 01 11 ± 04 10 ± 04 09 ± 02 GON-1 GON-2 GON-1 GON-2 GON-1 GON-2 GON-1 GON-2 GON-1 GON-2 156 07 ± 68 215 45 ± 70 160 37 ± 54 218 22 ± 00 164 53 ± 18 213 66 ± 79 169 56 ± 43 219 65 ± 20 160 53 ± 41 220 00 ± 99 08 ± 02 02 ± 02 06 ± 03 09 ± 00 03 ± 04 05 ± 04 09 ± 02 05 ± 05 08 ± 05 04 ± 02 69 ± 46 16 ± 53 −35 70 ± 76 −37 45 ± 55 −36 73 ± 35 −38 19 ± 93 −32 30 ± 18 −30 48 ± 23 −38 78 ± 51 −40 25 ± 98 −36 59 ± 11 −35 45 ± 12 −43 07 ± 18 −38 89 ± 56 40 82 ± 19 −37 56 ± 80 −44 85 ± 66 −40 46 ± 79 −36 04 ± 41 −34 70 ± 48 GON -1 GON -2 168 44 ± 74 220 25 ± 16 07 ± 02 09 ± 01 −35 66 ± 95 −32 29 ± 74 Dispersion time (min) 10 Delivered Publishing to: Rice University, Fondren Library rate, the biodisto recognize the position where FAbyattached to theTechnology surface affect particle size, the biodegradation IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 of the NPs FA bound with the gold probe is shown in tribution and physicochemical properties.35 36 Therefore, Copyright: American Scientific Publishers a surfactant-free nanoparticulate system has been sugFigure 4(g), in which the black spots (gold NPs) show the gested for preparation The GOCs are fortunately complace where FA attached to the surface of the NPs, conposed of components that possess surfactant properties for firming the presence of this ligand on the surface of GONs stabilization, which leads to spontaneously stabilized NPs HRTEM, which provided a closer view of FA-GONs on without the necessity for surfactant Therefore, the encapthe surface, showed the crystal lattice of the gold NPs sulation of PTX in the GONs is performed in appropriate (Fig 4(h)) The FA-GON had a mean diameter of approxconditions with high efficiency (Table III) The entrapimately 200 nm ment of the drug involves the incorporation of the drug PTX is one of the most effective anticancer agents into the hydrophobic cores of GONs due to hydrophoHowever, this drug has high incidences of adverse reacbic interactions, as previously shown in other research.37 38 tions, including neurotoxicity, myelosuppression and aller8 gic reactions Cremophor EL (polyethoxylated castor oil), Moreover, the solubility of PTX in the current drug-loaded the excipient incorporated for increasing drug solubilnano systems was increased compared to the pure drug ity, is also a cause to hamper the clinical usefulness of (Table III) PTX.15 Additionally, PTX is a hydrophobic drug with The in vitro release profile of PTX-loaded NPs with (PTX-FA-GON) or without FA (PTX-GON) was a poor aqueous solubility of approximately g/ml.33 investigated in PBS (pH 7.4) at 37 C (Fig 6) and comFor this reason, the controlled release behavior of PTX from polymeric NPs to suppress these adverse reactions pared with the pure drug The cumulative percentage has been highly recommended One common method to release from PTX-FA-GON and PTX-GON was approxiprepare drug-encapsulated NPs is the solvent evaporamately 55% and 45%, respectively, for days and reached tion method in which the polymers containing the drug 100% for both conjugates after month, which showed the are solubilized in one of the phases of an emulsion, and potential of the NPs as a controlled drug delivery system the polymer is precipitated to entrap the drug out of The lower cumulative release of PTX from PTX-FA-GON the solvent during solvent evaporation, which leaves the was perhaps due to the presence of the ligand on the NP drug-loaded particles suspended in the residual solvent.34 surface, which slightly increased the diffusion path On the other hand, drug release from Taxol® has been reported Generally, a surfactant must be used to make smallsized NPs in this emulsion solvent evaporation system, previously to be over 80% for PTX after 4–6 h.39 40 These results indicated that the GONs in this study could be but it can absorb to the NP surface to significantly J Biomed Nanotechnol 9, 1416–1431, 2013 1425 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Tran et al 100 GOC-1 1.5 80 % drug released 1.4 1.3 I1/I3 1.2 1.1 60 40 PTX-GON PTX-FA-GON pure drug 20 1.0 0.9 0.8 10 15 20 25 30 Time (day) 0.7 Figure Release profiles of PTX from NPs without and with FA 0.6 –4.0 –3.5 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0.0 logC FTIR analysis was used to investigate the molecular interaction between excipient and drug Figure shows the FTIR spectra of FA-GON, pure FA, pure PTX, PTX1.5 FA-GON and PTX-GON The FTIR spectrum of FA-GON 1.4 was observed to contain the bands of GOC and another peak at 1725 cm−1 , which indicated the presence of the 1.3 carboxylic group of FA This spectrum affirmed the con1.2 jugation of FA with GON in the preparation in which the carboxylic group of gelatin was activated by EDC/NHS 1.1 and reacted with the amine group of FA, leaving the carDelivered by Publishing Technology to: Rice University, Fondren Library 1.0 FA remaining The amide I band and N–H IP: 63.142.225.118 On: Sat, boxylic 10 Oct group 2015 of 07:05:21 bending vibration 0.9 Copyright: American Scientific Publishersof FA-GON also exposed a changed peak shape that moved slightly compared to the pure gelatin and 0.8 GOC The 900–700 cm−1 region characteristic of the bending regions of the functional groups of FA also appeared 0.7 in the FA-GON spectrum An N–H stretching of gelatin 0.6 at 3303 cm−1 was also observed for FA-GON Bands at 1338 and 1240 cm−1 indicated the in-phase combination of –4.0 –3.5 –3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0.0 C-N stretching, and a C O bending vibration appeared in logC the FA-GON spectrum but with a small changed position Figure Profile of intensity ratio (I1 /I3 from emission specfor FA-GON, implying a high net intensity of these bands tra against versus logC of GOC-1 and GOC-2 to determine the for FA-GON The FTIR results also indicated that there critical aggregation concentration was no interaction between the drug and GON or FA-GON, employed as long-circulating drug carriers to retard PTX as only drug peaks were observed Therefore, the drug was release over an extended period of time, especially for only presented in the NPs by encapsulation PTX-FA-GON, which is helpful for decreasing drug leakPXRD analysis was performed to identify the crystalline age from carriers before drug-loaded carriers arrive at the state of PTX in NP The disappearance of a sharp peak target cells.39 of PTX in both PTX-FA-GO and PTX-GO diffractogram I1/I3 GOC-2 Table III Loading content, encapsulation efficiency and solubility of paclitaxel at pH 7.4 (loading amount of drug is 10% compared to polymer amount), particle size and zeta potential of targeted functionalized NPs (FA-GON) and PTX-loaded NPs (PTX-GON and PTX-FA-GON) Solubility at 48 h (37 C) Samples FA-GON PTX-GON PTX-FA-GON 1426 Loading content (wt.%) Encapsulation efficiency (%) Drug-loaded nanoparticles ( g/mL) Pure drug ( g/mL) Particle size (nm) Polydispersity index (PI) Zeta potential (mV) 98 ± 22 13 ± 09 89 ± 2 91 ± 20 53 ± 15 16 08 ± 05 61 ± 07 253 69 ± 04 266 16 ± 89 285 31 ± 69 ± 03 08 ± 02 09 ± 04 −42 46 ± 19 −30 84 ± 78 −37 35 ± 87 J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery FA-GON 2840 2927 FA 1725 1262 1297 16461554 3002 PTX-FA-GON 1335 1689 PTX-FA-GON PTX-GON PTX-GON PTX pure PTX 4000 3000 2000 1000 Wavenumber (cm–1) 10 Figure FT-IR spectra of FA, FA-GON, pure PTX, PTX-loaded NPs without and with FA (PTX-GON and PTX-FA-GON) 20 30 40 50 60 theta Figure PXRD patterns of pure PTX, PTX-loaded NPs without and with FA (PTX-GON and PTX-FA-GON) indicates the reduction of drug crystallinity (Fig 8) This result can explain the reason why the drug dissolution of Delivered Technology to: Rice University, Library tial slightly increasedFondren the negative charge due to the carPTX could be enhanced comparedbytoPublishing the pure drug under IP: 63.142.225.118 On: Sat, 10 Oct 2015of07:05:21 boxylic groups FA Therefore, this result also affirmed the same conditions The release of PTX was controlled Copyright: American Scientific Publishers the aforementioned characterization With the incorpofor a long time for both PTX-FA-GON and PTX-GON, ration of PTX, the surface charges of PTX-GON and partly due to the enhanced solubility of PTX in these PTX-FA-GON were slightly decreased but still negatively nanopartilces (Table III) charged, which implied a good stability of the systems The particle size from DLS measurements showed The localization of or hydrophobic drugs, such as tamoxthat most of the GON-FA, PTX-GON and PTX-FA-GON ifen paclitaxel, within the micelle corona might shield the had an average diameter of approximately 250 nm, 260 nm surface charge by shifting the shear plane further from the and 280 nm, respectively (Table III), which is a some42 micelle surface, which leads to a reduced zeta potential what larger than the sizes observed in the TEM images These results are consistent with the results observed in the Biostability Study GONs and previous research.9 41 Drug-loaded NPs were an effective drug carrier only if they did not lose their In addition to checking the stability of the NPVs in aqueproperties at body temperature (37 C) and a physiologous systems as mentioned above, the stability of GON-2, ical pH (7.4) until the release of drug at the target site FA-GON, PTX-GON and PTX-FA-GON were tested in was achieved The particle size of the NPs after loading 100% human blood serum at 37 C to mimic physiological with PTX was increased, because the hydrophobic drug conditions The stability of self-assembled nanoparticulate entrapped in the hydrophobic core always creates a larger systems as drug carriers is essential for in vivo applicaparticle size than drug-free micelles.39 The PDI of FAtions of these systems Particle size values were monitored GON, PTX-GON and PTX-FA-GON were not above 0.15, as a function of the incubation time for 24 h to investigate which indicated a narrow particle size distribution of the the presence of particle aggregation (Fig 9) Compared to particles This same observation was obtained after storage the results observed in the aqueous conditions at pH 7.4, of month, which implied a stable nanoparticulate syswhich were to mimic the physiological condition (Tables II tem Table III also reveals the zeta potential of FA-GON, and III), the size of these NPs changed negligibly over the PTX-GON and PTX-FA-GON The surface charge of entire time-course of the test, which indicated an excellent FA-GON was approximately −42 mV, also indicated that stability and ultra-low fouling ability of the NPs However, the surface of the GONs stemmed primarily from the free the plots of FA-GON and PTX-FA-GON showed good carboxylic groups of gelatin and the presence of the FA stability throughout the whole time compared to the NPs without FA (GON-2 and PTX-GON) These results reinligand on the surface of the GONs because the zeta potenJ Biomed Nanotechnol 9, 1416–1431, 2013 1427 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Tran et al 24 hours 300 140 120 Cell viability (%) Particle size (nm) 280 260 240 220 PTX-GON PTX-FA-GON Taxol */# */# 100 */# 80 */# 60 */# # # 40 * 20 GON FA-GON PTX-GON PTX-FA-GON 200 */# * 0 2.5 180 25 250 2500 12500 25000 drugfree PTX concentration (ng/mL) 10 12 14 16 18 20 22 24 Incubation time (h) force the further application of these systems in in vivo experiments for the testing of their efficacy in the treatment of cancer However, the in vivo results would be expressed in another report PTX-GON PTX-FA-GON Taxol 160 140 Cell viability (%) Figure Stability of NPs with and without targeting ligand and those loaded with PTX in 100% human blood serum, continuously measured by DLS at 37 C (n = 3) 48 hours 180 120 */# */# 100 80 60 40 * ** * * 20 Cell viability (%) Delivered Cancer Cell Killing Effect by Publishing Technology to: Rice University, Fondren Library IP:of63.142.225.118 On: Sat, 10 Oct0 2015 07:05:21 Instead, cancer cell killing effect PTX-GON and PTX0 2.5 25 250 2500 12500 25000 drugCopyright: American Scientific Publishers free FA-GON was evaluated herein in comparison with Taxol® PTX concentration (ng/mL) (PTX formulated in Cremophor EL) The MTT assay was performed on PTX-GON and PTX-FA-GON for a compar72 hours 160 ison of their effects on the capability of anti-tumor treat* ment using the Hela cell line Cells were also incubated 140 PTX-GON PTX-FA-GON with empty NPs and the vehicle of Taxol® to ensure that the Taxol 120 cytotoxicity was caused by PTX itself or the carrier-bearing * 100 drug The range of concentrations of PTX chosen for the test corresponded to the plasma levels of the drug achiev80 able in humans.43 The vehicle of Taxol® and NPs chosen 60 for the testing of cytotoxic activity had the same concentration of Cremophor or blank NPs as the sample containing 40 ** the highest concentration of PTX (25,000 ng/ml) The cell */# 20 ** */# viability of Hela cells after treatment of PTX-GON, PTX0 FA-GON, Taxol® solution and vehicles after 24, 48 and 72 2.5 25 250 2500 12500 25000 drugh is shown in Figure 10 First, it should be emphasized free that no cytotoxicity activity was observed for the blank PTX concentration (ng/mL) NPs (without drug), even at a high concentration; the vehi*: Statistically significant difference, compared to Taxol® cle of Taxol® , Cremophor EL, which was absent in NPs, #: Statistically significant difference between PTX-GON and PTX-FA-GON showed a significant effect Therefore, the cytotoxicity of Figure 10 Viability of Hela cells incubated for 24, 48 and PTX-loaded NPs was only attributed to PTX, but the cyto® 72 hours with PTX-GON, PTX-FA-GON and Taxol® toxicity of Taxol was probably caused by Cremophor EL rather than PTX The cytotoxicity of Cremophor EL has from an inhibition of cell growth or from cytotoxicity The been reported previously.8 44 45 These results also suggest extent of cytotoxicity was influenced by both the concenthat GONs maintain the pharmacological activity of PTX tration and the incubation time, in which higher concenand efficiently deliver PTX to the cells The decrease in trations of PTX inhibited a greater amount of cells and a cell viability, as measured by the MTT test, can result 1428 J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Delivered by Publishing Technology to: Rice University, Fondren Library IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 Copyright: American Scientific Publishers Figure 11 Images of Hela cell viability upon the lowest concentration of PTX (2.5 ng/ml) at 72 hours under microscopy longer incubation period induced a larger number of cells to enter the G2 and M cell cycle phases Therefore, PTX was more active45 and led to a higher reduction in cell viability PTX encapsulated in both of the researched NPs, even the smallest concentration of PTX in the case of PTX-FA-GON, had a higher killing effect on tumor cells than Taxol® Figure 11 (supplementary data) indicates the more effective treatment of the NP through the appearance of cell images with the lowest concentration of PTX (2.5 ng/ml) at 72 h compared to the control image that contained culture media only Cells with the lowest PTX concentration of Taxol® showed almost the same viability as the control sample, but the cell viability in the images of PTXGON, PTX-FA-GON was very low The solubilization of PTX into NPs enhanced its in vitro anti-tumor activity compared to Taxol® , showing a smaller IC50 than Taxol® PTX entrapped in PTX-GON or PTX-FA-GON was readily available to interact with cancer cells and retained its cytotoxic activity At 48 h, the IC50 of Taxol® as a control was 30.07 ng/ml, but the cytotoxicity of PTX-FA-GON or J Biomed Nanotechnol 9, 1416–1431, 2013 PTX-GON was 0.54 ng/ml and 0.17 ng/ml, respectively Therefore, the results presented here are consistent with other published data that showed that the incorporation of PTX into NPs enhanced its anti-tumor activity compared to Taxol® because it showed smaller IC50 than Taxol® 46 47 CONCLUSION In summary, the current research investigated a novel synthetic method for the preparation of natural biocompatible GOCs The pH, temperature and amount of OA and gelatin were crucial for the formation of the self-assembled GONs, which were thermodynamically formed via selfassembly of the inner hydrophobic part of OA and the outer hydrophilic part of gelatin in an aqueous phase The NPs showed a uniform spherical shape and size and good stability under storage for at least month The newly synthesized GOC was also a good candidate for the encapsulation of a hydrophobic anti-cancer agent such as PTX, which not only enhanced drug dissolution but also controlled drug delivery This study also indicated that PTXloaded GO NPs, especially the NPs attaching the FA ligand 1429 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Tran et al on the surface, had a stronger cancer killing effect than Taxol® , even at a low drug concentration This result can be attributed to the effect of PTX because no cytotoxicity was observed with the current NPs, but Cremophor EL, the clinical vehicle of Taxol® , exhibited cytotoxicity Therefore, the NPs developed in this research could be considered an effective anti-cancer drug delivery system for cancer chemotherapy emulsion for lipophilic KW-3902, a newly synthesized adenosine A1 -receptor antagonist Chem Pharm Bull 50, 87 (2002) 13 A Ledo-Suárez, L Rodríguez-Sánchez, M C Blanco, and M A López-Quintela, Electrochemical synthesis and stabilization of cobalt nanoparticles, Physica Status Solidi a 203, 1234 (2006) 14 G Maxwell, Synthetic Nitrogen Products: A Practical Guide to the Products and Processes, Springer, New York (2004) 15 A K Singla, A Garg, and D Aggarwal, Paclitaxel and its formulations Int J Pharm 235, 179 (2002) 16 National Code of Practice for the Preparation of Material Safety Data Sheets, 2nd ed., [NOHSC:2011(2003)] Copyright ©Kilford & Acknowledgments: This work was supported by the Kilford Pty Ltd, October (2004) MEST for Human Resource Development Center for Eco17 K Alfred, G Carola, and B Simone, Molecular interactions in conjugates of dicarboxylic acids and amino acids J Mol Struct 661— nomic Region Leading Industry Project and by a grant 662, 239 (2003) from the Korean Health Technology R&D Project, Min18 Y.-Q Ye, F.-L Yang, F.-Q Hu, Y.-Z Du, H Yuan, and H.-Y istry for Health and Welfare, Korea (A092018) We would Yu, Core-modified chitosan-based polymeric micelles for controlled like to thank the Central Research Laboratory, Kangwon release of doxorubicin Int J Pharm 352, 294 (2008) 19 G A Digenis, T B Gold, and V P Shah, Cross-linking of gelatin National University for the use of the FTIR Finally, we capsules and its relevance to their in vitro-in vivo performance acknowledge KBSI (Chuncheon, Korea) for the TEM and J Pharm Sci 83, 915 (1994) particle size measurements 20 C Weber, C Coester, J Kreuter, and K Langer, Desolvation process and surface characterisation of protein nanoparticles Int J Pharm 194, 91 (2000) REFERENCES 21 Y B Patil, U S Toti, A Khdair, L Ma, and J Panyam, Single1 P A Maury, D N Reinhoudt, and J Huskens, Assembly of nanoparstep surface functionalization of polymeric nanoparticles for targeted ticles on patterned surfaces by noncovalent interactions, Current drug delivery Biomaterials 30, 859 (2009) Opinion in Colloid and amp Interface Science 13, 74 (2008) 22 V 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Eur J Pharm Biopharm 68, 112 (2008) Delivered by Publishing Technology to: Rice University, Fondren Library IP: 63.142.225.118 On: Sat, 10 Oct 2015 07:05:21 Copyright: American Scientific Publishers J Biomed Nanotechnol 9, 1416–1431, 2013 1431 ... powder was dried at room temperature under vacuum 1417 Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery Preparation of Self-Assembled. ..Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug Delivery hinders the inactivation of the encapsulated drug by prepotential... incubation with a 10 nm gold-labeled goat J Biomed Nanotechnol 9, 1416–1431, 2013 Tran et al Novel Multifunctional Biocompatible Gelatin-Oleic Acid Conjugate: Self-Assembled Nanoparticles for Drug