Gokhan
Yilmaz
abc,
Emine
Guler
d,
Caner
Geyik
e,
Bilal
Demir
d,
Melek
Ozkan
f,
Dilek
Odaci Demirkol
d,
Serdar
Ozcelik
f,
Suna
Timur
*dg and
C. Remzi
Becer
*c
aDepartment of Chemistry, University of Warwick, CV4 7AL, Coventry, UK
bDepartment of Basic Sciences, Turkish Military Academy, Ankara, Turkey
cPolymer Chemistry Laboratory, School of Engineering and Materials Science, Queen Mary, University of London, E1 4NS, London, UK. E-mail: r.becer@qmul.ac.uk
dDepartment of Biochemistry, Faculty of Science, Ege University, 35100, Bornova, Izmir, Turkey. E-mail: suna.timur@ege.edu.tr
eInstitute of Drug Abuse, Toxicology & Pharmaceutical Sciences, Ege University, 35100, Bornova, Izmir, Turkey
fDepartment of Chemistry, Faculty of Science, Izmir Institute of Technology, 35430, Urla, Izmir, Turkey
gCentral Research Testing and Analysis Laboratory Research and Application Center, Ege University, 35100, Bornova, Izmir, Turkey
First published on 18th October 2017
Over the past decade, there has been a great deal of interest in the integration of nanotechnology and carbohydrates. The advances in glyconanotechnology have allowed the creation of different bioactive glyconanostructures for different types of medical applications, especially for drug delivery and release systems. Therefore, the use of more efficient biocompatible nanocarriers with high loading capacity, low overall toxicity and receptor-mediated endocytosis specificity is still in focus for the enhancement of the therapeutic effect. Conjugation of sugar derivatives onto gold nanoparticles presents unique properties that include a wide array of assembling models and size-related electronic, magnetic and optical properties. Here, pH-responsive drug-conjugated glycopolymer-coated gold nanoparticles were prepared by functionalization of gold nanoparticles with thiol-terminated glycopolymers and then subsequent conjugation of doxorubicin (DOX). Among the four different glycopolymers, their drug release, physicochemical characterization (spectroscopy, particle size and surface charge) and in vitro bioapplications with four different cell lines were compared. As a result, pH-sensitive drug delivery via sugar-coated AuNPs was performed thanks to hydrazone linkages between glycopolymers and DOX. Comparative viability tests also demonstrated the efficiency of glycopolymer–DOX conjugates by fluorescence cell imaging. The obtained results reveal that AuNP homoglycopolymer DOX conjugates (P4D) have significant potential, especially in human neuroblastoma cells in comparison to cervical cancer cells and lung cancer cells.
Design, System, ApplicationGlycopolymer coated gold nanoparticles have been designed as pH responsive anticancer drug carriers through biological media and to release the drug under acidic conditions. Functional ‘Sweet or Sugar Coated Gold’ nanostructures were fabricated via functionalization of gold nanoparticles with thiol-terminated glycopolymers and then doxorubicin as an anticancer drug model was conjugated onto the surfaces via pH-sensitive hydrazone bonds. After characterization of the resulting nanoparticle drug conjugates, their cytotoxic properties and cell affinities towards various cancerous cell lines were evaluated. This design could be adapted to the controlled-release of various chemotherapeutics and these ‘Sweet nano structures’ could be applied for both imaging and therapeutic applications. |
Metal nanoparticle-based materials are appealing alternatives to fluorescence- or radiolabelled protein substrates, owing to their photo-stability, facile synthesis and ability to conjugate with biological molecules.11,12 Among these metallic nanoparticles, gold nanoparticles have paved the way thanks to the advantages such as chemical inertness, facile surface functionalization, amenable surface plasmon resonance and suitable optical properties for imaging.13–17
Concomitantly, there are other significant issues for NPs consisting of their stability and improvement of enhanced permeability and retention effect (EPR) without facing renal elimination and recognition by phagocytes.18 Hence, functionalization of AuNPs by constructing a core–shell architecture with polymeric layers including other recognition elements to facilitate the uptake of NPs has gained attention. To enable these specialties for AuNPs, glycofunctionalization has been a breakthrough both in surface modification for cell adhesion and potential anticancer activities applied with radiation therapy.19,20 Furthermore, prior to their modification on the surface of NPs, glyco-based structures as supramolecules were synthesized and used in bioapplications, successfully.21 The specific recognition properties of carbohydrates and unique physiochemical properties of gold nanoparticles are a desirable combination for the creation of multivalent nanoparticles for the treatment of chemotherapy/radiotherapy and fluorescence imaging. In our previous study, we presented polymethacrylic acid (PMAA) coated AuNPs and their post-modification with doxorubicin (DOX) to create a drug delivery system based on a pH-responsive manner.22 Instead of direct coating via thiol linkages, an additional polymer layer covered the surface of AuNPs before conjugation of DOX molecules and post-modified AuNP–PMAA particles via pH-sensitive hydrazone bonds.
Herein, we successfully synthesized mannose (Man) tagged AuNPs by covering the metallic core with methacrylic acid derivatives as a toolbox for pH-sensitive drug delivery. After defining bioconjugates in terms of size, surface charge and spectrophotometric properties, they were applied to four different cell lines including three cancerous and one healthy ones and their cytotoxic properties and cellular uptake were evaluated by fluorescence cell imaging. The efficacy of polymer-coated AuNPs with Man was also tested in a comprehensive comparison by using only polymer coated AuNPs without Man and free DOX.
As depicted in Fig. S1,† the new peak at 8.02 ppm appeared due to the formation of a triazole ring, which confirmed the click reaction between azide and alkyne groups. In addition, the existence of D-mannose C-1 peaks at 102.3 & 104.7 ppm revealed that the monomer is an anomeric mixture according to the 13C-NMR data. In the ESI-MS spectra, there is a clear peak at 387.4 m/z that corresponds to the molecular weight of the mannose glycomonomer with a theoretical mass of 387.2 m/z. The product was obtained as a white solid with a 44% yield. The main reason behind the low yield is the poor solubility of 3-azidopropyl methacrylate in the MeOH/H2O mixture which affects the efficiency of purification on the column.
Code | Composition | M n,Theo (g mol−1) | M n,GPC (g mol−1) | Đ |
---|---|---|---|---|
P1 | P((MAA)96) | 8400 | 12100 | 1.18 |
P2 | P((ManMac)46-r-(MAA)44) | 21800 | 24600 | 1.21 |
P3 | P((ManMac)45-r-(OEGMA)43) | 30500 | 29400 | 1.19 |
P4 | P((ManMac)94) | 37300 | 32700 | 1.21 |
The conversion values of monomers were calculated using the 1H NMR data by comparing the integrated signal intensity due to the aromatic protons of the RAFT agent at 7.8–7.9 ppm with the vinyl protons of monomers at 5.8–6.2 ppm. 1H NMR characterization revealed that quantitative conversions were achieved for each monomer. The Mn,SEC calculated by SEC measurement is slightly higher or lower than the theoretical molar mass mainly due to the different structures of the obtained polymers according to PMMA calibration standards. The theoretical number average molar mass of the synthesized polymers, Mn,Theo, was calculated by using the equation: Mn,Theo = ([M]0/[RAFT]0 × conversion × Mmon) + MRAFT, where [RAFT]0 is the initial RAFT agent concentration, [M]0 is the initial monomer concentration, Mmon is the monomer molecular weight, and MRAFT is the RAFT agent's molecular weight.
As depicted in Fig. 1 and S2,† no obvious changes in the SEC traces were observed and the Mn,SEC was exactly similar to that before treatment according to RI detection of SEC. However, variable wavelength detection of SEC at 308 nm did not show any significant peak after treatment suggesting that the polymer end groups are successfully modified to –SH. Moreover, the color of polymers which is due to the RAFT agent changed from pink to white after the successful cleavage of the RAFT end group.
Fig. 1 A) SEC analysis via RI detection; B) via VWD; C) 1H NMR characterization of the synthesized P2 homopolymer before and after reduction of the RAFT group. |
Polymer-substituted AuNPs were characterized in terms of size and zeta potential by using DLS. There were significant increases in the hydrodynamic volume between AuNPs and polymer–AuNPs, indicating the successful immobilization of the polymers onto the surface. As depicted in Fig. 2 and S3,† the size of the gold nanoparticles after coating with polymers increased from 55 ± 0.2 nm to 94 ± 0.7–101 ± 0.9 nm with the dispersity index (Đ) between 0.15 and 0.22, signifying a narrow particle size distribution. Furthermore, the negative zeta potential of AuNPs has changed depending on the nature of the polymer moieties. For instance, even though the magnitude of the negative zeta potential of P(MAA)-coated AuNPs increased from −26.9 ± 0.2 mV to −41.5 ± 2.3 mV due to the deprotonation of the carboxyl group (RCOOH ↔ RCOO− + H+) in aqueous solution, the negative zeta potential of P(ManMac)-coated AuNPs decreased to −24.0 ± 1.5 mV. According to the transmission electron microscopy (TEM) images (Fig. 2 and S6†), the size of the polymer-coated gold nanoparticles ranged between 57 ± 1.8 and 61 ± 1.7 nm. The average size of the monodisperse nanoparticles is summarized in Table 2.
Sample | DLS | TEM size (nm) | Zeta potential (mV) | SPRband(max) (nm) | |
---|---|---|---|---|---|
D h (nm) | Đ | ||||
AuNPs | 55 ± 0.2 | 0.11 | 43 ± 1.2 | −26.9 ± 0.2 | 519 |
AuNPs-P1 | 101 ± 0.9 | 0.15 | 57 ± 1.8 | −41.5 ± 2.3 | 521 |
AuNPs-P2 | 94 ± 0.4 | 0.21 | 59 ± 2.1 | −19.4 ± 7.3 | 522 |
AuNPs-P3 | 96 ± 0.8 | 0.22 | 61 ± 1.7 | −27.8 ± 2.8 | 524 |
AuNPs-P4 | 95 ± 0.6 | 0.18 | 60 ± 2.4 | −24.0 ± 1.5 | 523 |
In order to determine the amount of the polymer immobilized onto AuNPs, thermal gravimetric analysis (TGA) was utilized to analyze each material's thermal profile. As seen in Fig. 2 and S5,† each polymer-coated AuNP exhibited mass loss until approximately 650 °C due to the decomposition of the polymer onto the surface and the remaining fraction was the Au core of the synthesized nanoparticles which was unaffected even at temperatures as high as 650 °C. It was observed that polymer-substituted AuNPs contained approximately 86.2–88.7% polymer.
Initially, the binding capacity of each particle was determined via a DOX standard graph between 2.5–100 μM with the equation y = 0.006x − 0.009 (R2 = 9996). The DOX binding capacities of each conjugate are as follows: 41% for P1D, 25.6% for P2D, 32% for P3D, and 33.6% for P4D. As can be seen from the binding capacities, the structural changes of the polymer-coated AuNPs affected DOX binding in relation to the conjugation strategy. P1D, which has the most DOX amount, was obtained by the well-known EDC/NHS coupling under mild conditions due to the small functional moieties coming from MAA. Concomitantly, P2D, P3D and P4D conjugations were carried out by CDI coupling due to the urethane linkages which may be more sensitive with respect to EDC/NHS coupling. Moreover, this revealed the different Man amounts with more pendant –OH groups, which can be used effectively in the CDI method.
Particularly in P3D, which showed similar binding to P4D, there is less Man in comparison to OEGMA that contains additional –OH groups by inducing more DOX binding with respect to P2D conjugation. Afterwards, the same concentrations of DOX were utilized for the rest of the work. The spectrophotometric properties were investigated by presenting the normalized absorbance of each spectrum between 350–600 nm. According to Fig. 3, small shifts to 500 nm for the newly synthesized conjugates were observed relative to the maximum absorbance peak of DOX after conjugation. Additionally, these peaks are also beneficial to the subsequent drug release studies for measuring the absorbance of the release media.
Fig. 3 Normalized absorbance spectrum of AuNP/polymer–DOX core–shell particles and free DOX (all samples are at 10 μM DOX concentration). |
As the next characterization step, the hydrodynamic particle size distributions and surface charges of each bioconjugate were examined. DLS provided results in terms of RH, which is a hypothetical measure of the hydrodynamic radius of NPs in colloidal dispersions, to predict and control the fate of these NPs in cell media. Table 3 shows the differences in particle size and zeta potential between polymer coated particles and DOX-loaded bioconjugates. The DLS data confirmed that the DOX conjugated particles demonstrated the largest particle size distribution; the size of the negatively charged structure of Man tagged AuNPs decreased after bioconjugation.
Sample | Particle size (nm) | Zeta potential (mV) |
---|---|---|
P1D | 147 ± 25 | −22.7 ± 12 |
P2D | 239 ± 7.8 | −9.5 ± 0.9 |
P3D | 211 ± 27.1 | −12.3 ± 1.3 |
P4D | 171 ± 34.8 | −10.9 ± 0.5 |
Over the past decade, the advanced drug carrying nanotool called “trojan horse” has been developed with different strategies.24 In particular, pH-sensitive macromolecular polymeric structures,25 vesicular systems26 or metallic NPs27 were studied. Herein, Man tagged AuNP/polymer particles linked to DOX molecules with pH-sensitive hydrazone bonds were evaluated for their in vitro release profiles, in order to investigate the potential use of these particles as delivery carriers. One of the main characteristic features of tumor sites is that they have a slightly acidic microenvironment.28
Many of the pH-sensitive bonds between the drug and the carrier, which are formed via a typical hydrazone linkage, were evaluated at pH 5.3 as a model of the extracellular pH environment of cancer cells and at pH 7.4 as a model of the environment of healthy cells.29 As shown in Fig. 4, the drug release kinetics of DOX were much faster in an acidic pH environment (pH 5.3) than in neutral pH (pH 7.4) for all samples. DOX released from AuNPs was more controllable in comparison to free DOX, which reached 100% release after 72 h. On the other hand, under slightly alkaline conditions (pH 7.4) free DOX exhibited fast release while the cumulative release of conjugated particles was slow. In particular, a negligible amount of drug release from the P4D conjugate in physiological pH (pH 7.4) supports that it could be a good candidate as a pH responsive drug carrier. As a result, the slow drug release characteristics at neutral pH and increased drug release in acidic environments ensure AuNP–DOX bioconjugates to be promising drug carriers to improve intracellular drug release in the acidic microenvironment of most tumor types.
Fig. 4 Cumulative drug release graph of free DOX at pH 5.3 and pH 7.4 and AuNP/polymer–DOX bioconjugates at pH 5.3 and pH 7.4 for 72 h at 37 °C. |
Fig. 5 The effects of (A) AuNPs and (B) DOX conjugated AuNPs with free DOX on cell viability for 24 h [NT: non-treated cells. Bars represent ±SD, (n = 4)]. |
By using confocal laser microscopy, the DIC images were also included to monitor the morphology of neuroblastoma cells (SHSY-5Y) and were overlaid with the fluorescence images. As can be seen from Fig. 6, all samples are near, around and/or stacked on cell membranes after 30 min. Moreover, DOX and DOX conjugated glycopolymer coated AuNPs were found in the cytoplasm and interlocalized to the environment of the nucleus. This issue can be linked to the delivery of DOX due to the breakage of linkages in cytoplasmic media which can generally occur in endosomes after endosomal uptake of nanoparticles. In accordance with the in vitro drug release study, this successful experiment indicates that free DOX in cancer cell media, which have a lower pH than that in healthy cells, was spread more effectively. These data suggest that AuNPs are membrane permeable and have no effect on DOX sub-cellular localization even in concentrations under 1.0 μM DOX as the model cancer drug.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7me00086c |
This journal is © The Royal Society of Chemistry 2018 |