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Journal of Microencapsulation
Micro and Nano Carriers
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: https://www.tandfonline.com/loi/imnc20
Concanavaline A conjugated bacterial
polyester-based PHBHHx nanoparticles loaded with
curcumin for breast cancer therapy
Ebru Kilicay, Zeynep Karahaliloglu, Baki Hazer, Ishak Özel Tekin & Emir Baki
Denkbas
To cite this article:
Ebru Kilicay, Zeynep Karahaliloglu, Baki Hazer, Ishak Özel Tekin & Emir Baki
Denkbas (2016) Concanavaline A conjugated bacterial polyester-based PHBHHx nanoparticles
loaded with curcumin for breast cancer therapy, Journal of Microencapsulation, 33:3, 274-285, DOI:
10.3109/02652048.2016.1169325
To link to this article: https://doi.org/10.3109/02652048.2016.1169325
Published online: 06 Apr 2016.
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RESEARCH ARTICLE
Concanavaline A conjugated bacterial polyester-based PHBHHx nanoparticles
loaded with curcumin for breast cancer therapy
Ebru Kilicay
a,
Zeynep Karahaliloglu
b,
Baki Hazer
c,
Ishak €
Ozel Tekin
dand
Emir Baki Denkbas
ea
Zonguldak Vocational High School, B€ulent Ecevit University, Zonguldak, Turkey;bDepartment of Biology, Faculty of Science, Aksaray University, Aksaray, Turkey;cDepartment of Chemistry, Physical Chemistry Division, B€ulent Ecevit University, Zonguldak, Turkey;dDepartment of Medical Immunology, Faculty of Medicine, B€ulent Ecevit University, Zonguldak, Turkey;eDepartment of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey
ABSTRACT
The aim of this study was to evaluate therapeutic potential of curcumin-loaded poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBHHx nanoparticles (CUR-NPs) and concanavaline A conjugated curcumin-loaded NPs (ConA-CUR-NPs) for breast cancer treatment. The size and zeta potential of prepared NPs were about 228 ± 5 nm and 23.3 mV, respectively. The entrapment efficiencies of polymer/drug weight ratios, 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, ConA-1.25CUR-NPs, ConA-2.5CUR-NPs and ConA-5CUR-NPs were found to be68, 55, 45, 70, 60 and 51%, respectively. Optimized NPs formulations in the freeze-dried form were assessed with their short-term stability for 30 days of storage at 4C and 25C. Anticancer
activ-ity of ConA-CUR-NPs was proved by MTT assay and reconfirmed by double staining and flow cytometry results. The anticancer activity of ConA-CUR-NPs was measured in human breast cancer cells (MDA-MB 231) in vitro, and the results revealed that the ConA-CUR-NPs had better tumor cells decline activity.
ARTICLE HISTORY
Received 8 September 2015 Revised 25 February 2016 Accepted 17 March 2016 Published online 5 April 2016
KEYWORDS
PHBHHx; PHBHHx nanoparticles; curcumin; concanavaline A; drug delivery; human breast cancer cells
Introduction
Curcumin is a low molecular weight, hydrophobic, natural yellow polyphenolic compound extracted from turmeric, the rhizome of the herb Curcuma longa (Anand et al., 2008) and chemically named (diferuloylmethane)-(1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepadiene-3,5-dione) (Maheshwari et al.,2006). It is readily sol-uble in organic solvents such as acetone and ethanol. It shows extremely low solubility in aqueous solutions at acidic and physio-logical pH conditions, degradation at alkaline pH and poor bio-availability (Anand et al., 2007). It has been used in years for the treatment of many diseases containing neurological, cardiovascu-lar, neoplastic, pulmonary, and so on (Aggarwal and Harikumar,
2009). The most significant effect of curcumin is ability to suppress the proliferation of tumor cells. It has promising pharmaceutical properties including anti-oxidant (Ak and G€ulc¸in, 2008), anti-microbial (Mun et al.,2013), anti-inflammatory (Sandur et al.,2007), anti-fungal (Khalil et al., 2012), anti-depressant, wound healing, anti-carcinogenic activities (Verderio et al., 2013) and inducing apoptosis. It is also promising new natural compound for the treat-ment of several major human diseases, especially as a chemothera-peutic agent in a great variety of cancer models, containing ovarian, breast, head and neck, melanoma, colon, prostate and pancreatic cancers (Mukhopadhyay et al., 2001; Ak and G€ulc¸in,
2008; Aggarwal and Harikumar,2009). Despite these promising fea-tures, some properties, like hydrophobic inherent of curcumin, have limited its bioavailability and clinical application due to poor solubility, poor absorption and stability in aqueous systems, rapid metabolism and elimination (Mishra et al., 2008). Bioavailability of curcumin could be increased by encapsulating the drug in micro
or nanoparticle system in the form of polymeric drug carrier sys-tems (Shaikh et al.,2009).
Drug delivery systems are very interesting for clinical applica-tions. Many drug delivery tools have been developed as drug car-riers (Lavigne and Gorecki, 2006) to overcome these limitations. Among them, biomaterials from natural sources have been rapidly improved in the controlled drug release areas (Langer, 1990). Biodegradable polymeric nanoparticles are widely used to improve the therapeutic characteristics of different drugs. These carrier sys-tems improve drugs solubility, keep them from earlier degradation and advances controlled drug release and drug targeting (Khalil et al.,2013). They have excellent endocytosis and high encapsula-tion efficiency, passive tumour targeting delivery of a wide range of therapeutic agent (Brannon-Peppas and Blanchette, 2004). Among them, polyhydroxyalkanoates (PHAs) synthesized from renewable resources (Zinn et al.,2001) are promising materials for biomedical applications. They are natural, biodegradable and bio-compatible thermoplastics biomaterials (Lenz and Marchessault,
2005). There are different types of PHAs relating to the length of the side chain; short chain length (sclPHAs), medium chain length (mclPHAs) and long chain length (lclPHAs) hydroxyalkanoic acids. Those which, short chain length hydroxyalkanoic acids such as poly(3-hydroxybutyrate) (PHB) has biodegradable and biocompat-ible due to the fact that R-3-hydroxybutyric acid is a normal com-ponent of human blood (Wiggam et al., 1997) and tissue and located in the cell surrounding the eukaryotes (Reusch, 2000). The degradation product of PHB, 3-hydroxybutyric acid is non-toxic due to extensive intermediate product in all higher living being (Qu et al.,2005). However, it is too rigid, brittle and lack the super-ior mechanical properties using for biomedical applications (Hazer
CONTACTEmir Baki Denkbas¸ denkbas@hacettepe.edu.tr Department of Chemistry, Biochemistry Division, Hacettepe University, Beytepe 06800, Ankara, Turkey
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
VOL. 33, NO. 3, 274–285
and Steinb€uchel,2007). Thus, the physical and mechanical proper-ties of microbial polyesters need to be diversified and improved. mclPHAs copolymers have low melting point, low crystallinity and low mechanical properties for effectively using in biomedical appli-cations. To overcome these drawbacks, the different bacterial-based copolymers were synthesized to improve their mechanical and thermal properties. One of those, PHBHHx has improved mechanical characteristic and shows better elastic properties for using in controlled drug delivery systems, compared with poly(3-hydroxybutyrate), PHB (Hazer et al., 2012). PHBHHx has also improved hemocompatibility and cytocompatibility features. It has minor platelet adhesion, minimized hemolysis activity and erythro-cyte contact due to good biocompatible blood contact properties than PHB alone (Qu et al.,2006). PHBHHx has strong biocompati-bility for many types of cells, such as L929 (Wang et al., 2003), chondrocytes and bone marrow stromal cells compared with PHB (Qu et al., 2005). It had been proved that the main degradation products of PHBHHx, oligo(3-hydroxybutyrate-co-3-hydroxyhexa-noate) (OHBHHx), oligo(3-hydroxybutyrate)(OHB) and 3-hydroxybu-tyrate (3HB) were non-toxic to cultured cells in vitro (Cheng et al.,
2005; Sun et al.,2007; Zhao et al.,2007). Thus, PHBHHx was safely used as a biodegradable nanoparticle carrier for sustained release and hydrophobic drug delivery due to its degraded products are non-toxic and nonimmunogenic (Shan et al.,2006; Lu et al.,2010). Xiong et al. prepared and used hydrophobic drug rhodamine B isothiocyanate (RBITC)-loaded PHB, PHBHHx and PLA nanoparticles to investigate the intracellular release behaviours. The study results indicated that PHBHHx and PHB nanoparticles showed slower and longer intracellular sustained release rate than that of PLA nano-particles (Xiong et al.,2010).
In this study, hydrophobic poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate), PHBHHx copolymer was selected as a drug carrier tool (Kılıc¸ay et al., 2011) for hydrophobic drug, curcumin due to its ester backbone and alkane side chain. The benefit of using hydro-phobic nanoparticles was explained in the following: The accumu-lation of nanoparticles at the tumor site takes several hours. Thus, the nanoparticles get into the tumor site after the release of the drug is unwanted (Ma et al.,2012). PHBHHx is hydrophobic, which hinders the water diffusing into the nanoparticles and so prevents the fast release of the drug.
Controlled drug release formulation via ligand conjugated bio-degradable polymers-based nanoparticles has become one of the most important strategies in drug targeting. The ligand helps NPs to enter into cancer cells. Drug combination with ligand shows important efficacy compared to the free drug. Thus, the modifying of drug encapsulated nanoparticle surface with ligand could ensure significant benefit. The ligand, concanavaline A (ConA) affinities towards different carbohydrate is a lectin protein, extracted from the jack bean, Canavalia ensiformis. ConA is a pre-ferred ligand by reason of relatively good resistance to acidic pH and enzymatic degradation (Hurkat et al.,2012). Ligand immobil-ization could be realized by physical absorption, chemical and covalent binding. Covalent binding method forms covalent linkage between protein and functional groups on the particle surface by using EDC/NHS technique and is a simple and a common applica-tion (Tang et al., 2010). In this research, ConA conjugated CUR-loaded PHBHHx nanoparticles (ConA-CUR-NPs) were produced for targeted drug delivery to human breast cancer cells.
In the current study, first, we have developed curcumin-loaded PHBHHx NPs (CUR-NPs) to overcome the pharmacokinetic prob-lems. In our previous studies (Kılıc¸ay et al., 2011) demonstrated that PHBHHx micro and nanoparticles have been developed due to their great potential for having long circulation times. However, to the best of our knowledge, there has not yet been a showed
report about the use of PHBHHx nanoparticles as curcumin car-riers. Curcumin was successfully encapsulated into PHBHHx nano-particles and ligand, concanavaline A was conjugated by covalent bonding on the surface of these drug-loaded NPs.
This study aimed to investigate the short-term stability of nano-particles for demonstrating its suitability for administration and evaluate CUR-NPs and ConA conjugated CUR-NPs for use delivery carriers to control the release and increase the cellular uptake of curcumin. For this, we have prepared bare NPs, CUR-NPs and ConA-CUR-NPs with different amount of drugs and these samples were subjected to a series of characterization studies to assess their size and size distribution, zeta potential, morphology and structure. These techniques included zetasizer, zeta potential, scan-ning electron microscopy (SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). We have inves-tigated encapsulation efficiency and release profiles of CUR-NPs and ConA-CUR-NPs by using nanodrop. The short-term stability of all the prepared formulations in freeze-dried form were subjected to stability test by measurement of mean particle size, polydisper-sity index (PDI), zeta potential, encapsulation and loading effi-ciency for 30 days of storage at 4C and 25C, respectively. In
vitro tests were carried out on MDA-MB 231 to evaluate the cellu-lar uptake efficiency and potential endocytic mechanisms of the samples by double staining and flow cytometric assay method. Finally, we have investigated NPs cytotoxicity by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dipehnyl tetrazolium bromide (MTT) assay. The data demonstrated that CUR-NPs and especially, ConA-CUR-NPs show more effective than native drug against human breast carcinoma cells (MDA-MB 231).
Experimental
Materials
Poly (hydroxybutyrate-co-hydroxyhexanoate) PHBHHx [weight-aver-age molecular weight (Mw)¼ 834 000] containing 12 mol% of 3-hydroxyhexanoate (3-HHx) units was supplied by Procter & Gamble Company (Cincinnati, OH). Dichloromethane (DCM), curcumin (Mw ¼368.38) and Tween 80 were purchased from Merck (Darmstadt, Germany). Con-A (Canavalia ensiformis, type VI) and N-Hydroxysuccinimide (NHS) was obtained from Sigma-Aldrich (MO), ABD. N-ethylcarbodiimide hydrochloride (EDC-HCl) was obtained from AppliChem Biochemica GmbH (Darmstadt, Germany). All chemicals and solvent were used as received without further purifications.
Breast cancer cell line, MDA-MB 231 was obtained from American Type Culture Collection (Manassas, VA). Dulbecco modi-fied Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), tryp-sin-EDTA and 1% penicillin/streptomycin were purchased from Biological Industries, Israel. MTT (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) and live/dead cell double staining kit were obtained from Sigma-Aldrich.
Preparation of curcumin-loaded PHBHHx nanoparticles (CUR-NPs)
PHBHHx NPs were prepared by solvent evaporation technique based on our previous work (Kılıc¸ay et al., 2011). Tween 80 was chosen as surfactant during the nanoparticle preparation. The ionic surfactants showed important cytotoxicities, especially in case of cationic surfactants. It may occur the electrostatic interaction between charged groups of surfactants and the negatively charged of cell membrane (Hwang et al., 2015). Thus, it is of vital importance to use non-ionic surfactant. Tween 80 is a non-ionic
surfactant and commonly used as an aid of different kinds of hydrophobic compounds due to low toxicity and its significant influence on particle size and size distribution. To obtain CUR-NPs, PHBHHx and curcumin/ethanol (EtOH) solution with different poly-mer/drug weight ratios, 1/0.125 PHBHHx/CUR ¼1.25CUR-NPs; 1/ 0.25 PHBHHx/CUR ¼2.5CUR-NPs; 1/0.5 PHBHHx/CUR ¼5CUR-NPs respectively, were dissolved in DCM as an organic phase. Tween 80 and distilled water were prepared as an aqueous phase and then mixed with the organic phase, followed by homogenized using IKA T 125 Digital Ultra Turrax homogenizer. Mechanical stir-rer (Heidolph RZR 2021) and ultrasonic bath (Alex) were used to remove the organic solvent from the resulting emulsion. The formed CUR-NPs were liophilized (Christ, Germany) for the further characterization and applications.
Preparation of ConA-NPs and ConA-CUR-NPs
Covalent conjugation of ConA to free carboxyl groups on the par-ticles surface (ConA-NPs) were performed by coupling of carboxylic group of NPs and amine group of ConA using EDC/NHS as cou-pling reagent (Russell-Jones et al., 1999; Mo and Lim,2005; Misra and Sahoo, 2010). Briefly, 100 mg NPs were dispersed in PBS (pH 7.4), followed by the addition of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) (1 mg/ml) and then NHS (1 mg/ml). The resulting suspension was stirred at room tempera-ture by using rotatory at 30 rpm for two hours. Then the particles were centrifuged (Centrifuge 5810R) at 12 000 rpm for 20 min to remove the residual EDC and NHS. The activated nanoparticles were incubated for 2 h with the ligand, ConA. After incubation, the suspensions were centrifuged with PBS to remove the unconju-gated ConA trace and the supernatant was drawn to calculate the conjugation efficiency by nanodrop. The calculation of binding effi-ciency was done by subtracting the amount of ConA in the super-natant from the total amount of ConA used in conjugation reaction (conjugation efficiency¼ total amount of ConA-the amount of ConA in supernatant/total amount of ConA 100%). ConA-CUR-NPs were also prepared using the same method men-tioned above. The ligand was conjugated after the preparation of the drug-loaded nanoparticles (Liu et al.,2010). The obtained pre-cipitates were dried and then the existence of ConA on the NPs surface was confirmed by FTIR spectroscopy.
Characterization of nanoparticles
The morphology of NPs and CUR-NPs was observed using both SEM (Quanta FEG 450 model SEM) and AFM (Nanomagnetics, Turkey). The size and size distribution and zeta potentials of the nanoparticles produced at different parameters were determined by using zeta potential (Malvern Instruments, Model 3000 HSA, England). The effect of the ligand-coupling reaction to surface of PHBHHx nanoparticles were determined by FTIR (Perkin Elmer SpectrumOne, Nicolet 520, USA). The spectra were recorded over the range of 4000–500 cm1.
Drug encapsulation efficiency
Drug-loading and encapsulation efficiency of CUR-NPs (1.25 mg CUR; 2.5 mg CUR; 5 mg CUR) were measured by dispersion of nanoparticles from the aqueous nanoparticle suspension by centri-fugation at 12 000 rpm for 30 min. The precipitated nanoparticles were lyophilised and weighted. The amount of free curcumin in the yellowish supernatant and washed solutions collected was determined by nanodrop (Thermo Scientific Nano Drop 1000 Spectrophotometer) at a wavelength of 425 nm with a standard
curve (y¼ 0.0126x; R2 ¼ 0.9979). CUR-NPs were dissolved in dichlorometane and the amount of CUR in the particles was deter-mined. The loading and encapsulation efficiency (Anitha et al.,
2011; Liu et al., 2012; Anita et al.,2014) were calculated according to the following equations:
LE %ð Þ ¼ Total amount of curcumin the amount of free curcumin Weight of dried nanoparticles
100
EE %ð Þ ¼ Total amount of curcumin the amount of free curcumin Total amount of curcumin
100
The short-term stability study of nanoparticles
The physicochemical stability of nanoparticles was evaluated dur-ing storage its freeze-dried form at refrigerated condition (4C)
and the aqueous suspension at room temperature (25C) for
com-parative evaluation. One aliquot was freeze-dried for 24 h while the other one was kept at 25C. Aliquots were withdrawn at
pre-determined intervals (1 and 30 days) The physicochemical proper-ties including particle size, PDI, zeta potential, encapsulation and loading efficiency of all prepared nanoparticles were obtained from a total of 8 samples and evaluated by comparing the initial values. All experiments were carried out in triplicate for each sam-ple and the mean of four measurements was taken. The physico-chemical features for each sample in vitro were recorded as average values ± standard error deviation.
In vitro drug release profile
In vitro release profile of CUR from CUR-NPs and ConA-CUR-NPs including different quantities of CUR was studied using eppendorf method (Anita et al., 2014; Anitha et al., 2014). The experiments were realized in phosphate-buffered saline (PBS) of pH 7.4 at 37C at 150 rpm. Samples were incubated at 37C using a shaking
water bath (Heto SBD 50, Denmark). Six eppendorf tubes were used for all samples (1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, ConA-1.25CUR-NPs, ConA-2.5CUR-NPs and ConA-5CUR-NPs) at time inter-vals of 1, 4, 8, 16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448, 472, 496 and 520 h according to the sink condition. In each time point, 20 ll of this solution was drawn out and added 20 ll of ethanol to redissolved the released CUR. Then the samples were evaluated using nanodrop to deter-mine the quantity of the releasing curcumin. To calculate the amount of curcumin released from particles above mentioned, a standard of curcumin (0–160 lg/ml) was prepared and the absorb-ance was measured at 425 nm using nanodrop.
In vitro cytotoxicity study
The standard MTT assay was performed to determine bare NPs, CUR-NPs and ConA-CUR-NPs toxicity. MDA-MB 231 cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin mixture. Briefly, the cells were seeded in 96-well plates at 9 103
cells per well. Twenty-four hours later, the medium was removed and the cells were treated with a range of concentrations at 12.5, 25 and 50 lg/ml of CUR, bare NPs, CUR-NPs (1.25, 2.5 and 5 mg CUR-loaded nanoparticles) and ConA-CUR-NPs (1.25, 2.5 and 5 mg CUR-loaded ConA-conjugated nanoparticles).
The untreated cells were accepted as a control. After 24 h incuba-tion, 200 ll of 3 mg/ml MTT solution was added to each well and the plate was incubated at 37C, 5% CO
2 for 4 h. At the end of
exposure time, the medium was removed and isopropanol/HCl mixture added to each well and the absorbance was read at 570 nm by using a microplate reader (Biochrom Asys Expert Plus. Microplate Reader, Holliston, MA). Cell viability was calculated according to the following equation:
Cell viability %ð Þ ¼ A570ðsampleÞ A570ðcontrolÞ
100
Cell viability graph was drawn using the mean absorbance of three independent replicates.
Live/dead assay
The number of apoptotic and necrotic cells were determined using double staining method. MDA-MB 231 cells (15 103
cells per well) were grown in DMEM (high glucose) supplemented with 10% FBS and 1% penicillin-streptomycin at 37C, 5% CO
2. MDA-MB 231
cells were treated with different concentrations of free curcumin (25 and 50 lg/ml), bare NPs, CUR-NPs (drug-loading ratio; 1/0.5, 1/ 0.25, 1/0.125) and ConA-CUR-NPs (drug-loading ratio; 1/0.5, 1/0.25, 1/0.125) for 24-h period. The tissue culture plate (TCP) was taken as a control for double staining test. After 24 h incubation, the cell culture medium was removed and the cells were then stained by a mixture of calcein-AM and propidium iodide (PI) solution for 10 min. Survival rate were examined with Texas RedVR
and FITC fil-ters of Fluorescence Inverted Microscope (Leica, Germany). Calcein-AM only stains viable cells because itself is not a fluorescent mol-ecule and the calcein generated from Calcein-AM by esterase emits a strong green fluorescence. Propodium iodide is only able to enter through disordered areas of dead cell membrane and after intercalate with the DNA double helix, a red fluorescent sig-nal is produced. The survival rate of MDA-MB 231 cells treated with nanoparticles was determined by counting of live and dead cells from 6 randomly chosen microscopic fields.
Flow cytometric Annexin V-PI assay
MDA-MB 231 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37C, 5% CO2 on the
24-well TCP. After 24 h incubation, the culture medium was removed and 900 ll fresh medium was added first and then, the cells were treated with two different concentrations for all samples (25 and 50 lg/ml). After the sample loading, the volume of each well was completed to 1000 ll with medium. After 24-h incubation, the detached cells in the medium were collected, and the remaining
adherent cells were harvested by trypsinisation. After double wash-ing, PI and Annexin V were added in each sample according to the instructions of manufacturer. Cell death was measured by using flow cytometry (FC500 flow cytometer (Beckman-Coulter). In this assay, PI and Annexin V positive cells indicated late apoptosis or necrosis, and Annexin V positivity alone showed early apoptosis. In our study, early apoptotic cell ratio was small than 2% for each group. Therefore, we compared all dead cells for each group.
Statistical analysis
Numerical data were analysed using standard analysis of variance (ANOVA) techniques All experiments were completed in triplicate with three repeats for each experiment. Results were reported as average ± standard error of the mean.
Results and discussion
Preparation and size characterization of PHBHHx NPs, CUR-PHBHHx NPs and ConA-CUR-NPs
PHBHHx nanoparticles were prepared by solvent evaporation tech-nique described in our previous work (Kılıc¸ay et al.,2011). PHBHHx nanoparticles and CUR-loaded nanoparticles were characterized to evaluate the effect of the polymer, surfactant and drug amount and stirring rate on mean particle size, size distribution and surface charge (Table 1). In this study, Tween 80 was used as a surfactant. The existence of an anionic surfactant is significant to decrease the dynamic interfacial tension and to stabilize the nanoparticles. Tween 80 increased the steric repulsion between particles. Therefore particle size was decreased. The entrapment of drug into the nanoparticles caused increase of the particles size due to enlarge of the polymeric matrix. The enhancement of the amount of polymer caused a faster precipitation of the polymer during the preparation procedure. This effect caused the increase of nanopar-ticles size. The stirring rate is considerable parameters that effect size of the particles. The higher homogenization rate produces the smaller nanoparticles size. Increasing the homogenization rate lead to the enhanced energy conducted to the suspension medium and thus this effect caused a decrease in the nanoparticles size.
The size and polydispersity of NPs were determined by DLS.
Table 1 shows the size characteristics of the obtained
Table 1.Physicochemical properties of NPs and CUR-NPs. Polymer concentration (mg/ml DCM) Tween 80 concentra-tion (ml/ml) Curcumin concentration (mg/ml EtOHþ DCM) Homogenization rate
(% amplitude) Size (nm) Polydispersity index Zeta potential (mV) 10 0.06 – 90 440 ± 3 0.480 ± 0.018 27.5 ± 0.22 mV 6 0.06 – 90 327 ± 2 0.320 ± 0.043 24.2 ± 0.18 mV 2 0.06 – 90 228þ 5 0.299 ± 0.049 23.3 ± 0.09 mV 2 0.06 – 90 228 ± 5 0.299 ± 0.049 23.3 ± 0.11 mV 2 0.04 – 90 352 ± 10 0.340 ± 0.019 25.1 ± 0.27 mV 2 0.02 – 90 440 ± 15 0.485 ± 0.079 28.9 ± 0.33 mV 2 0.06 – 90 228 ± 5 0.299 ± 0.049 23.3 ± 0.20 mV 2 0.06 – 70 365 ± 12 0.369 ± 0.079 26.4 ± 0.35 mV 2 0.06 – 50 470 ± 11 0.601 ± 0.022 29.7 ± 0.41 mV 2 0.06 0.25 90 245 ± 5 0.310 ± 0.030 22.7 ± 0.13 mV 2 0.06 0.5 90 252 ± 4 0.312 ± 0.017 21.3 ± 0.02 mV 2 0.06 1 90 264 ± 3 0.317 ± 0.068 20.1 ± 0.10 mV Data represent mean ± SD, each sample was repeated in triplicate, n¼ 3.
Table 2. Characterization of surface functionalized nanoparticles.
Formulation Size (nm) PDI
Zeta potential (mV) Binding capacity (%) Control PHBHHx NPs 228 ± 5 0.299 ± 0.049 23.3 ± 0.24 – ConA-PHBHHx NPs 273 ± 8 0.310 ± 0.010 18.5 ± 0.11 90 ± 2
nanoparticles. PHBHHx NPs presented at the nanoscale with a nar-row size distributions. It was shown fromTable 1that the diameter was about 228 nm for blank NPs, 245 nm for 1.25CUR-NPs, 252 nm for 2.5CUR-NPs, 264 nm for 5CUR-NPs, respectively and polydisper-sity values were about 0.299, 0.310, 0.312 and 0.317, respectively. The optimized nanoparticle size was selected as a 228 nm for in vitro experiments. It was shown that the size of blank and CUR-NPs nanoparticles were slightly different. The diameters and poly-dispersity of CUR-NPs slightly increased due to entrapment of drug. The entrapment of the CUR did not affect significantly the physicochemical characteristics of the nanoparticles.
The zeta potential values of NPs and CUR-NPs were also showed in Table 1. Zeta potential reflects the surface charge of colloidal dispersions. It also controls dispersion stability. The zeta potential values of NPs, 1.25CUR-NPs, 2.5CUR-NPs and 5CUR-NPs were 23.3, 22.7, 21.3 and 20.1 mV respectively. A high potential value,23.3 mV supplies a high energy level barrier sta-bilizes the particles. The negative zeta potential was due to the existence of terminal carboxylic acid groups of the polymer pre-sent on the surface of particles. Electrostatic repulsion between particles with the same charge prevents the aggregation of the particles and provides extra stability (Zhao et al., 2012). It means that the high negative surface charge is a significant demonstra-tion for the colloidal system stability (Jeong et al., 2009; Shah et al.,2010).
The zeta potential of curcumin-loaded nanoparticles increased as the amount of curcumin increased. This is because that the drug adsorbed on the surface of nanoparticles caused masking effect on the terminal carboxylic groups and reduced the negative
zeta potential value (Masood et al., 2013). In all experiments, the optimum formulation, the particle size of 228 nm was used.
ConA was covalently connected to the carboxylic group of nanoparticles by EDC/NHS activation method. EDC comprises an active ester intermediate creates amide bond by reacting with ConA amine group. After ligand conjugation, the zeta potential formed less negatively charged. The average particle size of ConA-NPs found to be 273 ± 8 nm was in general slightly larger than the bare NPs. This surface ConA layer caused to increase of NPs size due to conjugation. The absolute value of z-potentials of ConA-NPs was found to be18.5 mV that was significantly increased as on coupling of ConA at the surface of NPs. This is because; posi-tively charged ConA that has amine group neutralizes negative charge of carboxylic group of polymeric nanoparticles during cova-lent bond forming (Hurkat et al., 2012). In the ligand attachment experiment, the binding capacity of ConA on the surface of NPs was found about 90% (Table 2). The ligand was successfully conju-gated onto the surface of NPs and CUR-NPs as schematically repre-sented in Figure 1(A). The optimized ConA concentration was selected at 0.25 lg/ll for all the experiment.
Morphology
As assessed by SEM and AFM, the morphology of NPs is generally spherical in shape with a relatively smooth surface, well separated and no aggregation was observed (Figure 1B). The mean diameters of NPs obtained by SEM were less than the values measured by DLS due to the dried nanoparticles dehydrated and shrunk. SEM images of 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, ConA-1.25CUR-NPs,
Figure 1.(A) Schematic representation of conjugation process of NPs and CUR-NPs with ConA, (B) The AFM and SEM image of PHBHHx NPs. PHBHHx NPs with a diam-eter 228 ± 5 nm was obtained. Scale bar is 2lm.
ConA-2.5CUR-NPs and ConA-5CUR-NPs did not very differ from those obtained for non-loaded PHBHHx NPs. They were relatively spherical shape (Figure 2A–F).
Surface chemistry analysis of NPs, CUR-NPs and ConA-CUR-NPs
Figure 3(A) shows the FT-IR spectrum of PHBHHx, PHBHHx NPs, CUR, 1.25CUR-NPs, 2.5CUR-NPs and 5CUR-NPs respectively. In the spectrum of PHBHHx NPs, the characteristic peaks were observed
Figure 2. SEM images of 1.25CUR-NPs (A), 2.5CUR-NPs (B), 5CUR-NPs (C), ConA-1.25CUR-NPs (D), ConA-2.5CUR-NPs (E), ConA-5CUR-NPs (F). Scale bars are 2 and 4lm.
Figure 3.(A) FTIR spectra of PHBHHx, PHBHHx NPs, curcumin, 1.25CUR-NPs, 2.5CUR-NPs and 5CUR-NPs and (B) of PHBHHx, PHBHHx NPs, ConA and ConA-PHBHHx NPs, respectively.
at 1720 cm1 and 3000 cm1 associated with carbonyl and OH stretch of PHB. Curcumin showed its characteristic absorption peaks at 3500 cm1, attributed to phenolic O-H stretching vibration and sharp absorption peaks at 1500 cm1 correspond to the –C¼ O and C–C, olefinic C-H vibrations, bands at 1250 and 1300 cm1 correspond to the aromatic –C–O stretching vibration. FTIR spectra confirmed the incorporation of curcumin in the CUR-NPs (Anitha et al.,2014).
Presence of ligands on the surface of nanoparticles was deter-mined by FTIR spectroscopy. The FTIR spectrum of ConA-NPs shows a peak of amide bond at 1500 cm1 due to conjugation between the carbonyl group of PHBHHx and the amino group of ConA (Khan et al.,2010; Hurkat et al.,2012) and shows characteris-tic bands at 1099 cm1, 1132 cm1, 1180 cm1 and 1228 cm1 respectively (Figure 3B).
Determination of drug content
Table 3 shows the entrapment (EE) and drug-loading efficiency (LE) of CUR-NPs and ConA-CUR-NPs, respectively. The EE and LE of 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, 1.25CUR-NPs, ConA-2.5CUR-NPs and ConA-5CUR-NPs were 68%, 55%, 45%, 70%, 60%, 51% and 16.8%, 17.7%, 33.6%, 15.7%, 17%, 30.2%, respectively (Table 3). It was seen that the high drug loading resulted in low drug encapsulation efficiency (Liu et al.,2010). When the drug con-centration was increased, the nanoparticulate system showed ten-dency to protrude the drug outside (Anitha et al.,2014).
The loading efficiencies of ConA-CUR-NPs were slightly decreased compared with CUR-NPs due to release of some drug adsorbed on the surface of NPs during washing steps in ligand attachment process (Misra and Sahoo,2010). As a result, curcumin is a hydrophobic drug and it was successfully entrapped into the hydrophobic core of PHBHHx.
Stability study
According to the stability results, the all samples stored at 25C
showed an important changes and instability after 15 days. There
occurred flocculation comparing with those stored at 4C and
thus, stability studies could not be continued. Therefore physico-chemical parameters were conducted only at 4C. The samples
showed narrow size distribution and a good homogeneity during storage at 4C, since the results demonstrated that the value of PI
was lower than 0.3 (narrow size distribution) in all nanoparticles. After freeze-drying, the particle size of the lyophilised NPs became a bit larger than that of their values prior to lyophilisation stored for 1 day. However, there was no important change in the mean size, PDI and zeta potential and the solutions stored for 1 day have almost similar mean particle size to those stored for 30 days at 4C.
The nanoparticles stored at 4C kept their starting morphology
during the period of 30 days. The size of particles showed initially a slight decreased and then a slight increased. The increasing of size may result partial aggregation among particles in process of time and also absence of a cryoprotectant. The slight decrease in EE and LE demonstrated that a small amount of drug expulsion and also minor ligand leakaged from drug-loaded and ligand con-jugated drug-loaded nanoparticles, respectively. The results were indicated inTable 4.
Consequently, no important changes in the mean particle size and size distribution, zeta potential, EE and LE of nanoparticles were observed for 30 days. According to the stability testing, all samples were found to be more stable under refrigerated condi-tions. These eight nanoparticles are very stable, indicate almost similar physicochemical stability and keep their integrity. Thus, it is the best way to lyophilise all the samples within 24 h for sustain-ing and keepsustain-ing the stability and integrity of nanoparticles.
In vitro drug release
In vitro release profiles of CUR-NPs and ConA-CUR-NPs are pre-sented inFigure 4. An initial burst observed within 4 min was 24% for 1.25CUR-NPs; 30% for 1.25ConA-CUR-NPs; 10% for 2.5CUR-NPs; 20% for 2.CUR-NPs; 5% for 5CUR-NPs and 7% for 5ConA-CUR-NPs followed by a prolonged release. This is probably a higher entrapment efficiency was associated to a higher burst effect and this fast release is also related to drug encapsulated near to nanoparticle surface. The drug-loading capacity of 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs, 1.25CUR-NPs, ConA-2.5CUR-NPs and ConA-5CUR-NPs are 16.8%, 17.7%, 33.6%, 15.7%, 17.0% and 30.2%, respectively. The higher polymer core nanopar-ticles were loaded, the more curcumin was released (Zhao et al.,
2012).
The release of curcumin reaches a plateau after 424 min for 1.25CUR-NPs and 1.25ConA-CUR-NPs; 472 min for 2.5CUR-NPs, 2.5ConA-CUR-NPs, 5CUR-NPs and 5ConA-CUR-NPs, 80% CUR from
Table 3. Entrapment efficiency (EE) and drug-loading efficiency (LE) with different drug concentration.
Samples EE (%) LE (%) 1.25CUR-NPs 68 ± 0.37 16.8 ± 0.11 2.5CUR-NPs 55 ± 0.25 17.7 ± 0.34 5CUR-NPs 45 ± 0.18 33.6 ± 0.27 ConA-1.25CUR-NPs 70 ± 0.30 15.7 ± 0.20 ConA-2.5CUR-NPs 60 ± 0.11 17.0 ± 0.09 ConA-5CUR-NPs 51 ± 0.19 30.2 ± 0.12 Data represent mean ± SD, n¼ 3.
Table 4. Mean diameter (nm), polydispersity index, zeta potential (mV), EE and LE of the nanoparticles after different periods of storage at 4C (data represent
mean ± SD).
4C
Bare NPs Size (nm) PD1 ZP (mV) EE % LE % 5CUR-NPs Size (nm) PD1 ZP (mV) EE % LE % Day1 230 ± 2 0.282 ± 0.020 25.2 ± 0.15 – – Day 1 269 ± 1 0.223 ± 0.010 23.6 ± 0.25 44.2 ± 0.15 33.0 ± 0.14 Day 30 233 ± 5 0.262 ± 0.05 25.9 ± 0.17 – – Day 30 270 ± 5 0.113 ± 0.045 23.4 ± 0.22 41.2 ± 0.16 29.2 ± 0.03 ConA-NPs ConA-1.25CUR-NPs Day 1 278 ± 5 0.237 ± 0.033 19.7 ± 0.21 Day 1 288 ± 3 0.211 ± 0.003 21.7 ± 0.17 69.0 ± 0.23 15.0 ± 0.11 Day 30 281 ± 3 0.266 ± 0.024 21.1 ± 0.34 Day 30 291 ± 2 0.208 ± 0.015 21.5 ± 0.19 67.4 ± 0.05 14.0 ± 0.04 1.25CUR-NPs ConA-2.5CUR-NPs Day 1 249 ± 2 0.267 ± 0.017 23.8 ± 0.23 67.0 ± 0.12 16.0 ± 0.08 Day 1 295 ± 1 0.218 ± 0.100 17.9 ± 0.23 59.8 ± 0.01 16.5 ± 0.10 Day 30 248 ± 3 0.249 ± 0.013 23.9 ± 0.22 62.0 ± 0.11 14.7 ± 0.05 Day 30 297 ± 4 0.217 ± 0.105 18.3 ± 0.37 57.0 ± 0.02 14.2 ± 0.02 2.5CUR-NPs ConA-5CUR-NPs Day 1 256 ± 2 0.211 ± 0.025 23.9 ± 0.13 54.4 ± 0.15 17.0 ± 0.08 Day 1 303 ± 1 0.225 ± 0.017 17.2 ± 0.17 50.6 ± 0.01 29.0 ± 0.05 Day 30 258 ± 2 0.022 ± 0.010 24.4 ± 0.17 50.2 ± 0.11 15.8 ± 0.01 Day 30 310 ± 8 0.106 ± 0.110 19.2 ± 0.10 46.5 ± 0.18 27.3 ± 0.03 Each sample was analysed in triplicate, n¼ 3.
1.25CUR-NPs and 84% CUR from 1.25ConA-CUR-NPs were released within 424 min and 70% CUR from 2.5CUR-NPs and 76% CUR from 2.5ConA-CUR-NPs; 44% CUR from 5CUR-NPs and 56% CUR from 5ConA-CUR-NPs were released within 472 min. These sustained and prolonged drug release originated from the drug diffusion localized in the PHBHHx core of the nanoparticles and matrix ero-sion mechanisms since CUR had very poor solubility in water, simi-lar to our previous release data for the different amount of
drug-loaded nanoparticles. Sustained release of CUR was observed from CUR-NPs and ConA-CUR-NPs over a period of 19 days. After bind-ing of ConA, the release rate was slightly increased due to the migration of CUR to the surface of the nanoparticles and the matrix integrities was decreased because of the conjugation reaction.
The sustained and controlled release of curcumin from CUR-NPs and ConA-CUR-CUR-NPs demonstrates an enough drug entrapment
Figure 4. In vitro release profile of curcumin from NPs/ConA-NPs in PBS solution (pH¼7.4) at 37C. Data are mean ± SD, n¼ 3.
Figure 5. In vitro cytotoxicity results (MTT assay) (A) for control, bare NPs and various concentrations of the free curcumin, (B) for 1.25CUR-NPs, 2.5CUR-NPs, 5CUR-NPs and (C) ConA-1.25CUR-NPs, ConA-2.5CUR-NPs, ConA-5CUR-NPs under 24 h of treatment. Data are expressed as means of a representative of three similar experiments.
within the nanoparticles. The results show that curcumin release from NPs was slightly slower as compared with ConA-CUR-NPs and PHBHHx nanoparticulate systems played important role in delaying curcumin release due to the hydrophobic interactions between the hydrophobic drug and hydrophobic polymer (Liu et al.,2000).
In vitro cytotoxicity study
Cytotoxicities of bare, CUR-NPs and ConA-CUR-NPs and also pure curcumin against MDA-MB 231 were evaluated by MTT assay (Figure 5). MDA-MB cells were exposed to different nanoparticle formulations for 24 h across a concentration range of 12.5–50 lg/l. Free curcumin showed a significant increase in cell mortality
compared to control and native PHBHHx nanoparticles. 5CUR-NPs treated MDA-MB cells showed 32% and 18% reduction in the cell viability compared to control and free curcumin, respectively (Figure 5B). After a 24-h incubation with CUR-NPs, MTT assay indi-cates dose-dependent decrease in cell viability. ConA-CUR-NPs demonstrated more cytotoxic effect on MDA-MB 231 cells than free curcumin and non-modified CUR-NPs. After exposure of breast cancer cell line to ConA-5CUR-NPs, cell viability showed reduction with 53% and 21% compared to control and 5CUR-NPs. The assay clearly demonstrated that ConA conjugation provide more effectiv-ity in arresting cell growth. The obtained data show that ConA-CUR-NPs have a marginal cytotoxic effect for breast cancer therapy.
Figure 6.(I) Fluorescent live/dead assay on MDA-MB 231 cells incubated with NPs by calcein-AM/PI double staining, values are mean ± SEM; n¼ 3 and (II) fluorescence microscopy images of MDA-MB 231 cells after 24-h incubation with nanoparticles. (A) Free curcumin, (B) 1.25CUR-NPs, (C) 2.5CUR-NPs, (D) 5CUR-NPs, (E) ConA-1.25CUR-NPs, (F) ConA-2.5CUR-ConA-1.25CUR-NPs, (G) ConA-5CUR-NPs. Scale bars are 100lm.
Udompornmongkol and Chiang reported that HCT116 (human colon carcinoma cell line) viability reduced when exposed to F-CUR (free curcumin) and F-CUR-NPs in the concentration range 1–50 mg/ml. Furthermore, CUR-NPs as potently as F-CUR decreased cell viability in a time- and dose-dependent manner (Udompornmongkol and Chiang, 2015). Similarly, Kumar et al. showed that C-PSA-NPs (curcumin-loaded PSA nanoparticles) had better uptake profile than curcumin in MCF-7 cell line (Kumar et al.,2014)
Live/dead assay
To evaluate whether the inhibition of cell viability by CUR-NPs and ConA-CUR-NPs, the survival rate was evaluated by calcein-AM/PI double staining. As shown inFigure 6I and II, more apoptotic cells were observed in cells treated with CUR-NPs and ConA-CUR-NPs. Especially, ConA-CUR-NPs showed cell viability with 4% when that of free curcumin was 17%. Furthermore, ConA-NPs and CUR-NPs showed a dose-related apoptotic response. 50 lg/ml concen-tration of ConA-1.25CUR-NPs was more active than 25 lg/ml con-centration one (Anand et al.,2010). The improved activity of ConA conjugated and CUR-NPs could be interpreted as better uptake and greater accumulation of nanoparticulate curcumin inside the tumour cells (Mohanty and Sahoo,2010).
In light of the previous finding that curcumin-loaded and lig-and-conjugated NPs showed a better apoptotic activity than curcu-min, our data indicate that ConA-CUR-NPs can be used as an efficient drug carrier for targeted killing of cancer cells.
Flow cytometric assay of cell viability using Annexin V and PI We used Annexin V and PI to investigate whether NPs induced apoptosis- or necrosis-mediated cell death. Figure 7 shows per-centage of dead cells due to both early and late stages of apop-tosis. Compared to free curcumin, curcumin-loaded and ligand-conjugated NPs decreased the late apoptosis and necrosis at 24 h.
Furthermore, a decrease in the number of late apoptotic and nec-rotic cells can be associated with the increased concentration of CUR-loaded in the NPs similar to live/dead assay results. Shi et al. reported the antitumor effects of concanavalin A (ConA) and Sophora flavescens lectin (SFL) on human breast carcinoma cells in vitro and in vivo. According the results, it was found that ConA and SFL exert anti-tumour actions against human breast carcinoma MCF-7 cells both in vitro and in vivo (Shi et al.,2014) These results indicates that the ConA-conjugation and CUR-loading induce both the apoptotic- and necrotic-mediated cell death.
Conclusions
CUR-loaded and ConA conjugated CUR-loaded PHBHHx NPs were prepared by solvent evaporation technique. All samples prepared have a spherical and smooth surface morphology with negative zeta potential, good stability and high drug encapsulation effi-ciency. Short-term stability studies showed that lyophilisation method seems to be a very suitable choice for preserving stability and maintained their integrity at 4C that it is of vital importance
for the controlled and targeted drug delivery. The release study showed an initial burst release and later sustained release of cur-cumin from CUR-NPs and ConA-CUR-NPs over a period of 19 days through in vitro release studies. The slightly higher drug release was observed with ConA-CUR-NPs than CUR-NPs due to the migra-tion of curcumin turn onto the surface of the nanoparticles during the ligand combining procedure. As a result, these nanoparticle systems significantly increased the circulation times of the drug. Furthermore, the cell line toxicity by MTT, cell survival and apop-tosis studies using double staining and flow cytometer assay showed that ConA-CUR-NPs had a high apoptotic activity and maximum localization into cell due to the targeted approach and so better uptake compared with NPs, CUR-NPs and free CUR.
As a result, ConA-CUR nanoparticulate system is a convenient and effective approach for the sustained and controlled release. Therefore, ConA-CUR-NPs are promising drug carriers to be used in
Figure 7. The curcumin-loaded and ligand-conjugated NPs induced cell death (apoptosis and necrosis) was measured by flow cytometry using Annexin V and PI staining. Values are mean ± SEM; n¼ 3.
targeted hydrophobic curcumin delivery applications and good therapeutic carrier tools for the treatment of breast cancer cell.
Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
Funding information
This work was financially supported by the Bulent Ecevit University for oral presentation in ICBN 2015: 17th International Conference on Biotechnology and Nanotechnology, 2015, Venice, Italy. Research Fund (Grant no. BEU-2015-YKD-33496813-01).
References
Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcu-min, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplas-tic diseases. Int J Biochem Cell Biol, 2009;41:40–59.
Ak T, G€ulc¸in I. Antioxidant and radical scavenging properties of curcumin. Chem Biol Interact, 2008;174:27–37.
Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: Problems and promises. Mol Pharm, 2007;4:807–18.
Anand P, Nair HB, Sung B, Kunnumakkara AB, Yadav VR, Tekmal RR, Aggarwal BB. Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bio-activity in vitro and superior bioavailability in vivo. Biochem Pharmacol, 2010;79:330–8.
Anand P, Sundaram C, Jhurani S, Ajaikumar B, Kunnumakkara AB, Aggarwal BB. Curcumin and cancer: An ‘‘old-age’’ disease with an ‘‘age-old’’ solution. Cancer Lett, 2008;267:133–64.
Anita A, Sreeranganathan M, Chennazhi KP, Lakshmanan VK, Jayakumar R. In vitro combinatorial anticancer effects of 5-fluo-rouracil and curcumin loaded N,O-carboxymethyl chitosan nano-particles toward colon cancer and in vivo pharmacokinetic studies. Eur J Pharm Biopharm, 2014;88:238–51.
Anitha A, Deepa N, Chennazhi KP, Lakshmanan VK, Jayakumar R. Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment. Biochimica et Biophysica Acta, 2014;1840:2730–43. Anitha A, Maya S, Deepa N, Chennazhi KP, Nair SV, Jayakumar R.
Curcumin-loaded N,O-carboxymethyl chitosan nanoparticles for cancer drug delivery. J Biomater Sci Polym Ed, 2011;23:1381–400.
Brannon-Peppas L, Blanchette JO. Nanoparticle and targeted sys-tems for cancer therapy. Adv Drug Deliv Rev, 2004;56:1649–59. Cheng S, Wu Q, Yang F, Xu M, Leski M, Chen GQ. Influence of
DL-beta-hydroxybutyric acid on cell proliferation and calcium influx. Biomacromolecules, 2005;6:593–7.
Hazer B, Steinb€uchel A. Increased diversification of polyhydroxyal-kanoates by modification reactions for industrial and medical applications. Appl Microbiol Biotechnol, 2007;74:1–12.
Hazer DB, Kılıc¸ay E, Hazer B. Poly(3-hydroxyalkanoate)s: Diversification and biomedical applications: A state of the art review. Mater Sci Eng C, 2012;32:637–47.
Hurkat P, Jain A, Jain A, Shilpi S, Gulbake A, Jain SK. Concanavalin A conjugated biodegradable nanoparticles for oral insulin deliv-ery. J Nanopart Res, 2012;14:1219.
Hwang TL, Alijuffali IA, Lin CF, Chang YT, Fang JY. Cationic addi-tives in nanosystems activate cytotoxicity and inflammatory
response of human neutrophils: Lipid nanoparticles versus poly-meric nanoparticles. Int J Nanomed, 2015;10:371–85.
Jeong I, Kim BS, Lee H, Lee KM, Shim I, Kang SK, Yin CS, Hahm DH. Prolonged analgesic effect of PLGA-encapsulated bee venom on formalin induced pain in rats. Int J Pharm, 2009;380:62–6. Khalil NM, Frabel do Nascimento TC, Casa DM, Dalmolin LF, de
Mattos AC, Hoss I, Romano MA, Mainardes RM. Pharmacokinetics of curcumin-loaded PLGA and PLGA-PEG blend nanoparticles after oral administration in rats. Colloids Surf B Biointerfaces, 2013;101:353–60.
Khalil OAK, de Faria Oliveira OMM, Vellosa JCR, de Quadros AU, Dalposso LM, Karam TK, Mainardes RM, Khalil NM. Curcumin antifungal and antioxidant activities are increased in the pres-ence of ascorbic acid. Food Chem, 2012;133:1001–5.
Khan TA, Husain Q, Naeem A. A novel and inexpensive procedure for the purification of concanavalin A from Jack Bean (Canavalia ensiformis) extract. Eur J App Sci, 2010;2(2):70–6.
Kılıc¸ay E, Demirbilek M, T€urk M, G€uven E, Hazer B, Denkbas EB. Preparation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHX) based nanoparticles for targeted cancer therapy. Eur J Pharma Sci, 2011;44:310–20.
Kumar SSD, Mahesh A, Mahadevan S, Mandal AB. Synthesis and characterization of curcumin loaded polymer/lipid based nano-particles and evaluation of their antitumor effects on MCF-7 cells. Biochim Biophys Acta, 2014;1840:1913–22.
Langer R. New methods of drug delivery. Science, 1990;249:1527–33.
Lavigne MD, Gorecki DC. Emerging vectors and targeting methods for nonviral gene therapy. Expert Opin Emerg Drugs, 2006;11:541–57.
Lenz RW, Marchessault RH. Bacterial polyesters: Biosynthesis, bio-degradable plastics and biotechnology. Biomacromolecules, 2005;6:1–8.
Liu J, Xu L, Liu C, Zhang D, Wang S, Deng Z, Lou W, Xu H, Bai Q, Ma J. Preparation and characterization of cationic curcumin nanoparticles for improvement of cellular uptake. Carbohydr Polym, 2012;90:16–22.
Liu M, Kono K, Frechet JMJ. Water-soluble dendritic unimolecular micelles: Their potential as drug delivery agents. J Control Release, 2000;65:121–31.
Liu Y, Li K, Liu B, Feng SS. A strategy for precision engineering of nanoparticles of biodegradable copolymers for quantitative con-trol of targeted drug delivery. Biomaterials, 2010;31:9145–55. Lu XY, Zhang Y, Wang L. Preparation and in vitro drug-release
behavior of 5-fluorouracil-loaded poly(hydroxybutyrateco-hydroxyhexanoate) nanoparticles and microparticles. J Appl Polym Sci, 2010;116:2944–50.
Ma Y, Sadoqi M, Shao J. Biodistribution of indocyanine green-loaded nanoparticles with surface modifications of PEG and folic acid. Int J Pharma, 2012;436:25–31.
Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple bio-logical activities of curcumin: A short review. Life Sci, 2006;78:2081–7.
Masood F, Chen P, Yasin T, Fatima N, Hasan F, Hameed A. Encapsulation of Ellipticine in poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) based nanoparticles and its in vitro application. Mater Sci Eng C Mater Biol Appl, 2013;33:1054–60.
Mishra VK, Mohammada G, Mishra SK. Down regulation of telomer-ase activity may enhanced by nanoparticle mediated curcumin delivery. Digest J Nanomater Biostruct, 2008;3:163–9.
Misra R, Sahoo SK. Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy. Eur J Pharm Sci, 2010;39:152–63.
Mo Y, Lim LY. Preparation and in vitro anticancer activity of wheat germ agglutinin (WGA)-conjugated PLGA nanoparticles loaded with paclitaxel and isopropyl myristate. J Cont Rel, 2005;107:30–42.
Mohanty C, Sahoo SK. The in vitro stability and in vivo pharmaco-kinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials, 2010;31:6597–611.
Mukhopadhyay A, Bueso-Ramos C, Chatterjee D, Pantazis P, Aggarwal BB. Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines. Oncogene, 2001;20:7597–609.
Mun SH, Joung DK, Kim YS, Kang OH, Kim SB, Seo YS, Kim YC, Lee DS, Shin DW, Kweon KT, et al. Synergistic antibacterial effect of curcumin against methicillin-resistant Staphylococcus aureus. Phytomedicine, 2013;20:714–18.
Qu XH, Wu Q, Liang J, Qu X, Wang SG, Chen GQ. Enhanced vascu-lar-related cellular affinity on surface modified copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx). Biomaterials, 2005;26:6991–7001.
Qu XH, Wu Q, Chen GQ. In vitro study on hemocompatibility and cytocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexa-noate). J Biomater Sci Polymer Edn, 2006;17(10):1107–21. Reusch RN. Transmembrane ion transport by polyphosphate/poly
(R)-3-hydroxybutyrate complexes. Biochemistry, 2000;65(3):280–95.
Russell-Jones GJ, Veitch H, Arthur L. Lectin-mediated transport of nanoparticles across Caco-2 and OK cells. Int J Pharm, 1999;190:165–74.
Sandur SK, Pandey MK, Sung B, Ahn KS, Murakami A, Sethi G, Limtrakul P, Madmaev V, Aggarwal BB. Curcumin, demethoxy-curcumin, bisdemethoxydemethoxy-curcumin, tetrahydrocurcumin and tur-merones differentially regulate antiinflammatory and anti-proliferative responses through a ROS-independent mechanism. Carcinogenesis, 2007;28:1765–73.
Shah M, Naseer MI, Choi MH, Kim MO, Yoon SC. Amphiphilic PHA-mPEG copolymeric nanocontainers for drug delivery: Preparation, characterization and in vitro evaluation. Int J Pharm, 2010;400:165–75.
Shaikh J, Ankola DD, Beniwal V, Singh MN, Kumar D. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. Eur J Pharm Sci, 2009;37:223–30. Shan C, Qiong W, Yan Z, Bing Z, Qiang CG. Effect of
poly(hydroxy-butyrate-co hydroxyhexanoate) microparticles on growth of
murine fibroblast L929 cells. Polym Degrad Stabil, 2006;91: 3191–6.
Shi Z, Chen J, Li CY, An N, Wang ZJ, Yang SL, Huang KF, Bao JK. Antitumor effects of concanavalin A and Sophora flavescens lec-tin in vitro and in vivo. Acta Pharmacologica Sinica, 2014;35:248–56.
Sun J, Dai ZW, Zhao Y, Chen GQ. In vitro effect of oligo-hydroxyal-kanoates on the growth of mouse fibroblast cell line L929. Biomaterials, 2007;28:3896–903.
Tang J, Liu Y, Yin P, Yao G, Yan G, Deng C, Zhang X. Concanavalin A-immobilized magnetic nanoparticles for select-ive enrichment of glycoproteins and application to glycopro-teomics in hepatocelluar carcinoma cell line. Proteomics, 2010;10:2000–14.
Udompornmongkol P, Chiang BH. Curcumin-loaded polymeric nanoparticles for enhanced anti-colorectal cancer applications. J Biomater Appl, 2015;30:537–46.
Verderio P, Bonetti P, Colombo M, Pandolfi L, Prosperi D. Intracellular drug release from curcumin-loaded PLGA nanopar-ticles induces G2/M block in breast cancer cells. Biomacromolecules, 2013;14:672–82.
Wang YW, Wu Q, Chen GQ. Reduced mouse cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating. Biomaterials, 2003;24:4621–9.
Wiggam MI, O’Kane MJ, Harper R, Atkinson AB, Hadden DR, Trimble ER, Bell PM. Treatment of diabetic ketoacidosis using normalization of blood 3-hydroxybutyrate concentration as the endpoint of emergency management. A randomized controlled study. Diabetes Care, 1997;20(9):1347–52.
Xiong YC, Yao YC, Zhan XY, Chen GQ. Application of polyhydrox-yalkanoates nanoparticles as intracellular sustained drug-release vectors. J Biomater Sci, 2010;21:127–40.
Zhao P, Wang H, Yu M, Liao Z, Wang X, Zhang F, Ji W, Wu B, Han J, Zhang H, et al. Paclitaxel loaded folic acid targeted nanopar-ticles of mixed lipid-shell and polymer-core: In vitro and in vivo evaluation. Eur J Pharm Biopharm, 2012;81:248–56.
Zhao Y, Zou B, Shi ZY, Wu Q, Chen GQ. The effect of 3-hydroxybu-tyrate on the in vitro differentiation of murine osteoblast MC3T3-E1 and in vivo bone formation in ovariectomized rats. Biomaterials, 2007;28:3063–73.
Zinn M, Witholt B, Egli T. Occurrence, synthesis and medical appli-cation of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev, 2001;53:5–21.
