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Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry
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Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry Gema Marcelo ∗ , Ekrem Kaplan 1 , M. Pilar Tarazona, Francisco Mendicuti ∗ Departamento de Química Analítica, Química Física e Ingeniería Química, Universidad de Alcalá, Alcalá de Henares 28871, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 29 November 2014 Received in revised form 21 January 2015 Accepted 23 January 2015 Available online xxx Keywords: Catechol Cyclodextrin Gold nanoparticles Doxorubicin Drug release Polymer
a b s t r a c t A copolymer containing -cyclodextrin, catechol and polyethylene glycol groups in its side chain was designed for the in situ synthesis and coating of gold nanoparticles (Au@PEG–CD NPs). These platforms were designed as a smart carrier and traceable delivery probe of the chemotherapeutic Doxorubicin drug (Dox). The coated polymer forms stable complexes with Dox in water with a high binding constant (K = 2.3 × 104 M−1 at 25 ◦ C), which is one hundred times greater than those reported for its complexation with native CD. Therefore, Au@PEG–CD NPs were able to load 0.01 mg of the drug per mg of NP and to release up to 60% of it in 48 h at 37 ◦ C. In addition, Au@PEG–CD NPs had the capacity to act as a quencher of Dox fluorescence when it was complexed with CD in the NP organic shell. This feature allows the Dox release to be tracked by monitoring the recovery of its fluorescence in real time. Therefore, the Dox release kinetics and the influence of temperature on the thermal stability of Dox/CD complexes on Au@PEG–CD NP were investigated. The increase in temperature favors the dissociation of the complexes and subsequent Dox release from the NP. The first order rate constant for drug releasing was 1.1 × 10−2 min−1 with a half-life time of 63 min at 37 ◦ C. Finally, the great potential of the carrier/probe double nature of Au@PEG–CD NPs was demonstrated in real time inside HeLa cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Dox) is a chemotherapeutic drug used in the treatment of a wide variety of cancer types. As with many other drugs, its low water solubility and poor photostability are drawbacks that need to be solved along with the side effects related to its citotoxicity. In order to overcome these limitations Dox has been introduced in different carriers [1–4]. Among these different vectors, gold nanoparticles (Au NPs) are receiving great attention because of their relatively easy surface functionalization [5] and adequate cell penetration [6]. Moreover, Au NPs are known for their optical properties derived from the localized surface plasmon resonance (LSPR), which have a strong impact on a wide range of bioapplications such as imaging, sensing and photothermal therapy [7–9]. An interesting feature is related to the effects that Au NPs induce on organic dye fluorescence properties [10–12]. Au NPs have mostly been reported to act as a dye fluorescence quencher. However, this ability is strongly dependent on the Au NP-dye distance, among other
∗ Corresponding authors. Tel.: +34 918854672. E-mail addresses: [email protected] (G. Marcelo), [email protected] (F. Mendicuti). 1 Current address: Istanbul Technical University, Science and Letters Faculty, Chemistry Department, 34469 Maslak, Istanbul, Turkey.
factors [12]. This behavior renders Au NPs great potential to be used as probes in the monitoring of different biomedical processes such as drug delivery and the determination of key molecules associated to important diseases. For example, Wang et al. [13] and El-Sayed et al. [14] described how Au NPs that bind Dox with an acid-labile linkage, allow the release of Dox into the cell acid lysosomes to be tracked by a recovery of Dox fluorescence after the breaking of the linkage. In another work, Lee et al. used Au NPs functionalized with Cy5.5 to monitor the activity of a protease involved in cancer in vivo. Upon interaction with the protease, the linkage between Cy5.5 and the Au NP breaks off, leading to the recovering of the Cy5.5 fluorescence [15]. On the other hand, cyclodextrins (CDs) are natural cyclic oligosaccharides formed of glucopyranose units. They possess a basket-shaped topology with an inner cavity which exhibits relatively hydrophobic behavior. CDs are able to form reversible, non-covalent inclusion complexes in aqueous media with a wide variety of hydrophobic guests with dimensions that fit inside their cavities. Complexation favors guest solubilization and stability allowing CDs to be used as drug carrier/delivery systems [16]. In addition, complexation processes are usually accompanied by a negative enthalpy change [17], and consequently the amount of the drug released could be increased with temperature, as well as the rate of the process. Another advantage of using CDs as
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carriers is the fact that the transported drug does not need to be chemically modified. Some works dealing with the modification of gold nanoparticles with CDs to transport hydrophobic drugs have been reported [5,18–20], but none of them explores their capability of acting as a probe to obtain information in ‘real time’ about the drug carrier/delivery process. The aim of the present work consisted in designing and preparing surface modified Au NPs with an oligomer which contains -cyclodextrin (CD) in its structure. Subsequently, these hybrid gold–CD NPs (Au@PEG–CD NPs) were investigated as Dox carrier/delivery systems by initially analyzing their cargo capacity and the time required for its release with temperature. The ability of the gold core to act as a quencher of Dox fluorescence was exploited to obtain information about the stability of the drug:CD on gold NP surface complexes, as well as its rate kinetics of release. In addition, the Dox–plasmon interaction allowed us to follow the Dox carrier and subsequent delivery inside the HeLa cells by fluorescence imaging. 2. Materials and methods 2.1. Materials -Cyclodextrin (-CD) (≥98%), p-toluenesulfonyl chloride (99%), sodium azide (99.5%), calcium hydride (CaH2 , 97%), triphenylphosphine (99%), pyridine (99%, ACS reagent), acetone (99.5%, ACS reagent), N,N-dimethylformamide (DMF) (99.8%, ACS reagent), dimethyl sulfoxide (DMSO), borax (Na2 B4 O7 ·10H2 O), magnesium sulfate (MgSO4 ), 2,2 -azobisisobutyronitrile (AIBN), poly(ethylene glycol) methyl ether methacrylate (Mn = 300), methacryloyl chloride, Doxorubicin hydrochloride (Dox) (98.0–102.0%), triethylamine (TEA), chlorhydric acid (HCl) and sodium hydroxide (NaOH) were all purchased from Sigma–Aldrich and used as received. Ethyl acetate (EtAc) was purchased from Scharlau. Hydrogen tetrachloroaurate (III) (HAuCl4 ) and dopamine hydrochloride were obtained from Alfa Aesar. A 0.01 M phosphate buffered saline solution (PBS) of pH = 7.4 (NaCl 0.138 M; KCl – 0.0027 M) (Sigma–Aldrich) was used as a buffer.
2.5. Synthesis of the PEG–catechol–CD polymer A -cyclodextrin monomer (0.5 g), a catechol monomer (0.24 g) and a poly(ethylene glycol) methyl ether methacrylate (2.0 g) were dissolved in 8 mL of oxygen-free DMSO and heated at 83 ◦ C. Azobisisobutyronitrile (AIBN; 6.5 mg) was added in nitrogen atmosphere and the reaction mixture was stirred at 83 ◦ C for 24 h. After that, 50 mL of water (pH = 6) were added to the reaction mixture while stirring. The solution was centrifuged and the supernatant was dialyzed (pore size: 2000 MWCO) for five days. After that, the water was evaporated and the molecular weight of the resulting polymer was characterized by 1 H NMR and UV–vis spectroscopy. The 1 H NMR spectrum of the PEG–catechol–CD oligomer in DMSO is shown in Figure 2S (Supporting Information). 1 H NMR (300 MHz, DMSO-d6), ı (ppm): 6.67 (d, 1H, Aryl-H), 6.64 (s, 1H Aryl-H), 6.53 (d, 1H, Aryl-H), 3.34, 5.78–5.63 (m, 14H in CD), 4.90–4.85 (m, 7H in CD), 4.50–4.45 (m, 6H in CD), 3.66–3.54 (m, 28H in CD), 3.42–3.24 (overlap with HDO, m, 16H in CD), 4.2–3.8 (s, 3H final methyl group in polyethylene glycol chain) and 1.0–0.6 (CH2 and CH of polymer backbone). The mole proportion of each unit in the final structure of the oligomer was calculated taking into account the integral areas of the following signals: the signals between 6.3 and 6.8 ppm for 3H of the catechol aromatic ring, the signal between 6.5 and 6.0 ppm which was assigned to 14H belonging to the CD and the signal between 3.8 and 4.3 ppm corresponding to 3H of the end methyl group in the polyethylene glycol chain. Due to the interaction of the polymer with the columns, the use of the GPC technique to determine the molecular weights was unsuccessful. Therefore, the polymer molecular weight was estimated by UV–vis spectroscopy [24]. First, for a water solution of polymer of well-known concentration in g/L, the molar concentration of the polymer was calculated by using the Beer–Lambert law by using the molar absorptivity of dopamine hydrochloride (2560 M−1 cm−1 in water at 280 nm). Then by combining the concentration (g/L) and the molar concentration (M) it was possible to estimate the molecular weight.
2.2. Synthesis of a methacrylate monomer functionalized with a catechol group
2.6. Determination of the association constants for the Dox with PEG–catechol–CD polymer complexation
The synthesis and NMR characterization of a methacrylate monomer functionalized with a catechol group were performed according to what was described elsewhere [21].
For the 1:1 stoichiometry of the Dox:PolCD (where PolCD means PEG–catechol–CD polymer) complexation process which is described by the following equilibrium,
2.3. Synthesis of mono-6-amino-6-deoxy-cyclodextrin (NH2 -CD)
Dox + PolCD Dox : PolCD
(1)
the association constant K is written as, The synthesis and NMR characterization were carried out as described elsewhere [22]. 2.4. Synthesis of a methacrylate monomer functionalized with ˇ-CD The synthesis and NMR characterization of a methacrylate monomer were performed according to what was reported [23].
I = I0 + (I∞ − I0 )
(1 + K[Dox]0 + K[PolCD]0 ) −
K=
[Dox : PolCD] [Dox][PolCD]
(2)
where [PolCD], [Dox] and [Dox:PolCD] are the concentrations of the PEG–catechol–CD (per mol of CD), Dox and the 1:1 complex respectively. By assuming two fluorescent species at the equilibrium, the free Dox and the complexed one, Dox:PolCD, the binding constants can be determined from the non-linear dependence on fluorescence intensity with the total initial PEG–catechol–CD concentration, [PolCD]0 , according to the following expression:
2 (1 + K[Dox]0 + K[PolCD]0 ) − 4K 2 [Dox]0 [PolCD]0 2K[Dox]0
(3)
where I, I0 and I∞ are the corrected fluorescence intensity at each [PolCD]0 , for the free Dox (in the absence of a PEG–catechol–CD) and for the complex (at [PolCD]0 →∞) respectively. [Dox]0 and [PolCD]0 are the initial concentrations of the Please cite this article in press as: G. Marcelo, et al., Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.01.041
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drug guest (=5 × 10−7 M) and the PEG–catechol–CD derivative. Eq. (3) assumes that the Dox fluorescence intensity is quenched upon PEG–catechol–CD addition. Protocols: fluorescence spectra for a set of solutions, named A, of PEG–catechol–CDs with concentrations increasing up to 14 mg/mL in the presence of a constant concentration of Dox (5 × 10−7 M) in a PBS buffer, and for another set, named B, with exactly the same PEG–catechol–CD concentrations but in the absence of Dox, were recorded. In order to remove the strong scattering on emission due to polymer aggregation with the concentration, each emission spectrum was corrected by subtracting at each spectrum of set A the corresponding one of set B. The Dox/PEG–catechol–CD PBS solution emission spectra after subtraction are shown in Figure 3S (Supporting Information). 2.7. In situ formation and surface modification of gold nanoparticles with PEG–catechol–CD 150 mg of PEG–catechol–CD were dissolved in 17 mL of MiliQ water. Then 1 mL of HAuCl4 water solution (16 mg/mL) was added under magnetic stirring (300 rpm). Direct observation of the Au NP formation took place after 3 min, the solution became blue. The mixtures were left stirring for 20 min. The resulting particles were cleaned by redispersion in distilled water by the use of an ultrasound bath (Elma). After that, they were stirred for 10 min and finally they were centrifuged. This process was repeated three times. 2.8. Determination of amount of Dox adsorbed on Au@PEG–CD NPs Dry Au@PEG–CD NPs were re-dispersed in a Dox PBS solution (2.5 × 10−5 M). After that, the colloidal dispersion was left stirring overnight. The amount of Dox loaded in the Au@PEG–CD NPs was obtained by UV–vis spectroscopy. To do this, the absorbance of the initial Dox solution without gold nanoparticles and the absorbance of the final supernatant solution after isolating the gold nanoparticle by centrifugation (12,000 rpm) were determined. The extinction coefficient was 11,500 cm−1 M−1 [25]. 2.9. The kinetics of Dox release Two samples of Au@PEG–CD NPs (8 mg) were re-dispersed in either 23 mL of Dox 1.0 × 10−6 M (in PBS) and 4.3 mL of Dox 2.2 × 10−5 M (in PBS). Colloid dispersions were left stirring overnight. After that, NPs were isolated by centrifugation. The amount of Dox adsorbed on NPs was determined by UV–vis spectroscopy. The Dox concentration in each one was determined to be 1.0 × 10−6 M and 1.1 × 10−5 M. Then, they were re-dispersed in 4.3 mL of fresh PBS to obtain two stock colloidal dispersions which were stored at 4 ◦ C. To study the kinetics of Dox release, an aliquot of both stock colloidal dispersions was re-dispersed in a fresh PBS buffer. Thus, the final Dox concentrations in the colloidal dispersion were 3.3 × 10−8 M and 3.6 × 10−7 M (0.06 mg/mL of Au@PEG–CD NPs in PBS) and the steady-state fluorescence spectrum was tracked with time. The experimental time depended on the temperature of the experiments. Spectra were collected until the fluorescence intensity remained constant. The experimental times were 510, 450 and 410 min at 25, 37 and 45 ◦ C respectively. 2.10. Cell growth and interaction with the [Au@PEG–CD NPs. . .Dox] complexes HeLa cells (ATCC CCL-2) were kindly served by the cells culture unit of UAH. Cells were cultured with an RPMI1640 medium containing 10% fetal bovine serum (FBS, Sigma Ref. F7524) and
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10% antibiotic antimycotic solution (Sigma, Ref. A5955) at 37 ◦ C under 5% CO2 , 95% air-humidified atmosphere. The culture media was changed every 2 days. After that, HeLa cells were seeded into 60 -Dish 35 mm glass bottom (Ibidi, Ref. 81158) at an initial cell density of 2 × 104 cells/dish. After 48 h, 50 L of PBS containing Au@PEG–CD NPs (2 mg/mL) complexed with Dox at two different concentrations, 2.8 × 10−6 and 8.2 × 10−5 M, were added and the cells were incubated. Cells were imaged at different incubation times depending on the system.
3. Results and discussion Throughout the different sections of this manuscript, we will initially explain the design, synthesis and characterization of the polymer containing CDs which will be used to both prepare and modify the Au NPs surface. In addition, the thermodynamics of the complexation process in aqueous medium between this polymer and Dox will be studied. Subsequently the strategy for both the preparation and the modification of Au NPs with the polymer will be described and the carrier capability, its interaction with Dox and the kinetics of the drug release will be investigated. In the last part, the drug-carrier interaction will be analyzed in a more realistic system after its uptake in HeLa cells.
3.1. Preparation and characterization of a cyclodextrin polymer (PEG–catechol–CD) A (PEG–catechol–CD) copolymer was synthesized by the radical polymerization reaction of poly(ethylene glycol) methyl ether methacrylate and two methacrylamide derivatives containing cyclodextrin (CD) and catechol moieties respectively (structures of the monomers are depicted in Figure S1 of the Supporting Information). Each monomer plays an important role in the function of the final colloid system: the CD macroring from the first monomer provides an effective site for the drug interaction, the PEG gives both hydrophilic behavior and biocompatibility [26] to the system and the catechol groups from the third monomer are reducing agents versus HAuCl4 allowing for the formation of Au NPs [27,28]. Scheme 1 shows the chemical structure of the PEG–catechol–CD copolymer. 1 HNMR together with UV–vis spectroscopies allowed us to characterize the copolymer. The composition of the side chain was: 6.5 mol % CD, 14.7 mol % catechol groups and 78.8 mol % of poly(ethylene glycol) chains. The molecular weight was estimated to be 6000 g mol−1 . Therefore, each oligomer is composed on average of 12 units containing poly(ethylene glycol), two units bearing catechol groups and one with a pendant CD macrocycle. The water behavior of the PEG–catechol–CD copolymer was analyzed by DLS. The variation of the hydrodynamic diameter with the polymer concentration at 25 ◦ C is depicted in Fig. 1. The PEG–catechol–CD copolymer exhibits a strong capacity to form aggregates above a 7 g/L concentration. Its hydrodynamic diameter is greater with the increase of polymer concentration, reaching 120 nm for a polymer concentration of 100 g/L. We explained the aggregation behavior with the increase in polymer concentration considering the moderate hydrophobicity of catechol groups [29]. We consider that catechol groups buried inside aggregates via hydrophobic and – stacking interactions form an inner core, while the highly hydrophilic PEG chains, as an outer shell layer, surround it. FE-SEM characterization for a PEG–catechol–CD water solution (16 mg/mL) shows spherical aggregates of polymer with a diameter ranging between 100 and 150 nm. This high value in relation to the hydrodynamic diameter could be due to an aggregation process during the water evaporation on the carbon grid used for
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Scheme 1. Chemical structure of PEG–catechol–CD copolymer and its schematic representation.
Fig. 1. (A) Hydrodynamic diameter as a function of polymer concentration. (B) FE-SEM of a dried drop of polymer solution (16 mg/mL).
the SEM characterization. Nevertheless, it could be indicative of the presence of self-assembling polymer structures.
3.2. Interaction of the PEG–catechol–CD with Dox The complexation process of Dox with the PEG–catechol–CD polymer illustrated in Fig. 2 (top) was studied in a PBS buffer solution by using fluorescence spectroscopy. The Dox concentration was fixed at 5 × 10−7 M and the PEG–catechol–CD concentration was increased up to 14 g/mL. Dox concentration was sufficiently low to avoid dimer formation. The complexation of Dox with the PEG–catechol–CD is characterized by some changes in the photophysical properties of Dox. A decrease in its corrected fluorescence intensity and an increase in its weighted average fluorescence lifetimes, , obtained from fluorescence decay profiles (equation 2S supporting information) were observed. Changes are attributed to the Dox inclusion into CD cavities. Some preliminary experiments on Dox in the presence of a PEG–catechol polymer which lack CDs, exhibit not changes in the fluorescence intensity with concentration. The changes of both properties for Dox at different PEG–catechol–CD concentrations are depicted in Fig. 2(A) and (B) respectively. The variation of the fluorescence intensity with polymer concentration allows us to determine the binding constant for the Dox complexation with PEG–catechol–CD. The curve depicted in Fig. 2(A), obtained by adjusting the experimental data from steadystate measurements to the proper equation for a 1:1 Dox:CD
Fig. 2. Top: Illustration of the complexation process between the PEG–catechol–CD and Dox. (A) Corrected fluorescence intensity measured at 590 nm (exc = 480 nm) and (B) weighted average lifetime for Dox at [Dox]0 = 5 × 10−7 M and different concentrations of PEG–catechol–CD (PolCD) (expressed in CD molar concentration) measured at 600 nm upon excitation wavelength of 360 nm.
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Fig. 3. Top: Illustration of the redox reaction that leads to the in situ formation and functionalization of gold nanoparticles with a coating of PEGMA–catechol–CD. (A) FE-SEM image of Au@PEG–CD NPs. (B) UV–vis–NIR absorption spectrum of Au@PEG–CD NPs.
stoichiometry (Eq. (3)) shows a reasonable fit. This provided a binding constant of (2.3 ± 1.7) × 104 M−1 at 25 ◦ C. The curve depicted for the variation in Fig. 2(B) was also obtained by fitting the experimental data to an equation similar to 3 (see Supporting Information, equation 3S), but using instead of the fluorescence intensity and the K value obtained from steady-state fluorescence intensity measurements. The value binding constant of 2.3 ×104 M−1 is two magnitude orders higher than the one obtained for the Dox complex with native CD at the same temperature. The incorporation of CDs in a polymeric structure leads to a cooperative inclusion behavior enhancing the binding affinity of CDs with a broad spectrum of drugs [30,31]. For instance, a binding constant of 3.7 × 104 M−1 was reported between the chemotherapeutic drug albendazole and a block copolymer modified with CDs. This value was 23 times higher than that obtained for the albendazole complexation with the native CD [31]. According to this result, the PEG–catechol–CD could be considered an excellent system to modify the Au NPs in order to obtain a stable carrier for Dox.
were determined in water by UV–vis–NIR spectroscopy, exhibiting the LSPR [32] located at 650 nm. Moreover, two more bands centered at 285 and 400 nm, as Fig. 3(B) depicts, were also observed. The higher energy band corresponds to the catechol groups and the other one was attributed to a catechol oxidation product formed during the redox reaction [33–35]. To complete the characterization, thermo gravimetric analysis allowed us to determine the weight percent of the organic coating in the Au@PEG–CD NPs that was 25 wt %. Therefore, there was 0.25 mg of polymer in 1 mg of Au@PEG–CD NPs. The number of CD moles was estimated to be 4.7 × 10−8 per mg of Au@PEG–CD NPs. 3.4. Interaction of Au@PEG–CD NPs with Dox and the kinetics of Dox release The potential of Au@PEG–CD NPs to act as a Dox carrier was evaluated in terms of their drug loading capability and
3.3. Preparation of gold nanoparticles and functionalization with a PEG–catechol–CD coating It has been reported that catechol groups suffer an oxidative self-conversion into their quinone forms by releasing protons and electrons under mild reductive conditions [27,28]. They can then be used for the gold salt precursor reduction to form Au NPs. Therefore, the Au NPs modified with PEG–catechol–CD copolymer were prepared in situ by stirring during 20 min a water solution of PEG–catechol–CD copolymer in the presence of an excess of HAuCl4 . After 4 min, the solution gradually turned blue. The resulting particles were named Au@PEG–CD NPs. See the scheme in Fig. 3 (Top). FE-SEM of Au@PEG–CD NPs of Fig. 3(A) showed gold nanoparticles with a size of ca. 90 nm. A thick shell of PEG–catechol–CD polymer covers the Au NP. Optical properties for Au@PEG–CD NPs
Fig. 4. Quantification of the release of Dox from Au@PEG–CD NPs in a PBS buffer solution with time at two temperatures: 25 ◦ C () and 37 ◦ C ( ).
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Fig. 5. Top: Illustration of the influence of the temperature in both the Dox delivery and trigger ON of its fluorescence. (A) Raw fluorescence emission spectra with time of Au@PEG–CD NPs loaded with Dox (7 × 10−7 M) in PBS at 25 ◦ C, (exc = 480 nm). (B) Fitting of experimental fluorescence data to a first-order kinetic reaction at 25 (), 37 ( ) and 45 ◦ C ( ).
subsequent release with time feasibility. The estimated maximum amount of Dox complexed with the Au@PEG–CD NPs (Au@PEG–CD NPs. . .Dox) was 0.01 mg per mg of NPs (1.7 × 10−8 moles per mg of NPs). According to the CD mole content determined above, this value indicates that Dox is adsorbed on Au@PEG–CD NPs in nearly an equimolar concentration in relation to CD. The amount of Dox released at different times was determined at two temperatures, 25 and 37 ◦ C. We assume that the re-dispersion of (Au@PEG–CD NPs. . .Dox) in a fresh PBS buffer leads to a de-complexation process, in which the maximum Dox amount freed corresponds to its equilibrium concentration. Therefore, at equilibrium, Dox can either appear as free Dox or as complexed (Au@PEG–CD NPs. . .Dox) forms. Fig. 4 shows the percentage of drug released with time which, as expected, depends strongly on the temperature. After 48 h, this amount reached 23% at 25 ◦ C, but at 37 ◦ C it was 60%. This result can be explained considering that the Au@PEG–CD NPs. . .Dox complexation process was governed enthalpically. As a result, the complex stability constant diminishes with temperature [19,20].
As stated earlier in this paper, when a chromophore molecule close to the gold NP surface interacts with the gold NP plasmon field, it results in a quenching of its fluorescence [15,36]. Therefore, the recovery of fluorescence upon Dox release from Au@PEG–CD NPs can be a useful tool to obtain information in real time about the thermal stability of the Dox. . .CD complexes on the carrier and to follow the kinetics of drug release in solution. Steady-state fluorescence intensity was used to monitor the release with time of Dox from Au@PEG–CD NPs for two colloidal solutions of (Au@PEG–CD NPs. . .Dox) in PBS containing Dox at two different concentrations 3.3 × 10−8 M and 3.6 × 10−7 M at three temperatures: 25, 37 and 45 ◦ C. For the colloidal solution with Dox at 3.3 × 10−8 M, Dox fluorescence was initially not detected and even with the recorded time no emission was observed for any of the temperatures. This might be because of either the very low Dox concentration or the overlapping of its emission with the NPs dispersion scattering band. However, an initial emission of Dox was observed together with the background NPs scattering for the colloidal solution containing Dox at a concentration of 3.6 × 10−7 M.
Fig. 6. Images of fluorescence (in red) obtained with Laser scanning confocal microscopy for the [Au@PEG–CD NPs. . .Dox] dispersion in a PBS buffer at 37 ◦ C after 30 min (left) and 180 min (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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The variation of the emission band with time at the three temperatures was studied. As an example, Fig. 5(A) depicts the emission spectra changes at 25 ◦ C. There is an increment in intensity of the Dox emission band with time at any temperature, which is attributed to the increase in the amount of fluorescent free Dox. Releasing kinetics was quantified at these temperatures by adjusting the experimental data to first-order reactions. Rate constant values were 7.9 × 10−3 , 1.1 × 10−2 and 2.2 × 10−2 min−1 with half-life times (time in which the 50% of Dox is released) of 88, 63 and 32 min at 26, 37 and 45 ◦ C respectively. The decomplexation process is therefore thermally favored and the delivery rate increases with temperature. Evident information of the Dox release at 37 ◦ C was obtained by laser scanning confocal microscopy upon monitoring the images of a dispersion of (Au@PEG–CD NPs. . .Dox) in a PBS buffer (2 mg/mL of Au@PEG–CD NPs with a concentration of Dox of 2 × 10−6 M) at two different times, 30 min and 180 min. As Fig. 6 shows, a significant enhancement of Dox fluorescence emission intensity after 180 min was observed, which is due to the release from Au@PEG–CD NPs. 3.5. Interaction of (Au@PEG–CD NPs. . .Dox) with HeLa cells Most of the time, drug delivery from the carrier occurs with hardly noticeable changes of the fluorescent properties neither in the carrier nor the drug [37]. Only very few studies, where the drug release leads to changes in the drug fluorescence intensities and lifetimes, were reported [3,13,14]. In this next section, we demonstrate that Au@PEG–CD NPs can act simultaneously as an effective cellular carrier and as a probe to track in real time the Dox cellular release in a living system by monitoring the increment in its fluorescence intensity. HeLa cells were incubated at 37 ◦ C with (Au@PEG–CD NPs. . .Dox) containing Dox at two different, 7 × 10−8 and 2 × 10−6 M, concentrations. These concentrations were not very different from those used in the previous steady-state fluorescence experiments of the preceding section. The cellular uptake of (Au@PEG–CD NPs. . .Dox) at 30 min and 240 min of incubation time was investigated by recording CLSM images. For a Dox concentration of 7 × 10−8 M there is no Dox fluorescence emission after 30 min of incubation (data not shown). Dox is hardly released from Au@PEG–CD NPs at this time and its emission is totally quenched. However, as depicted in Fig. 7, after 240 min of incubation, a weak fluorescence emission of Dox was detected in the cell cytoplasm. Therefore, even at the low concentration used, the experiments enable us to corroborate that the Au NP also acts as a very
Fig. 7. Laser scanning confocal microscopy images of HeLa cells treated with Au@PEG–CD NPs. . .Dox containing a [Dox] = 7 × 10−8 M after 240 min.
sensitive probe to study the drug releasing in cells by turning on its fluorescence upon delivery. For a Dox concentration of 2 × 10−6 M after 30 min of incubation, as depicted in Fig. 8, the Dox fluorescence emission can be perfectly observed in the cytoplasm of HeLa cells. At this Dox concentration, according to the kinetics of release, only 63 min is necessary for a 50% of Dox release at 37 ◦ C. The Dox emission intensity in the inner cells increases considerably after 240 min of incubation, namely after four times the half-life time. This fact could be attributed mainly to a greater amount of Dox released from the carrier together with a greater internalization of the (Au@PEG–CD NPs. . .Dox) complexes. HeLa cells incubated with only the Dox drug at 6.0 × 10−7 M presented a high degree of internalization (see Supporting Information, Figure S4). The fluorescence of Dox was observed in the cytoplasm as well in the nuclei at 240 min of incubation. Free Dox is known to be transported into cells via diffusion [4]. The difference in the localization and internalization degree of free Dox compared with when it is transported as (Au@PEG–CD NPs. . .Dox) could be attributed to the different cellular uptake mechanisms.
Fig. 8. Laser scanning confocal microscopy images of HeLa cells treated with Au@PEG–CD NPs. . .Dox containing a [Dox] = 2 × 10−6 M after different incubation times: (A) 30 min and (B) 240 min. Scale bar 50 m.
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4. Conclusions Au NPs modified with a polymeric shell containing CDs were prepared, Au@PEG–CD NPs. Their capability to act simultaneously as both a Dox carrier and a probe to monitor the Dox–CD interaction on the carrier was demonstrated in solution and in a living system as well. The preparation of these gold nanoparticles was carried out in two steps: (i) synthesis of a copolymer which contains catechol, CD and PEG groups that present a remarkable high binding affinity toward Dox (K = 2.3 × 104 M−1 at 25 ◦ C); and (ii) in situ formation and decoration of gold nanoparticles with the copolymer by using the catechol redox chemistry. 1 mg of Au@PEG–CD NPs was able to complex 0.01 mg of Dox and to release it in a 60% after 48 h at 37 ◦ C. The interaction between Dox and the LSPR led to the quenching of Dox fluorescence. Therefore, by analyzing the recovery of Dox fluorescence after its delivery, quantitative information about the Dox release kinetics was obtained. An increase in the temperature contributed notably to the dissociation of the Au@PEG–CD NPs. . .Dox complex and subsequent drug release. To conclude, the success of the double nature carrier/probe was demonstrated in a living system in real time. Dox is transported to the cytoplasm of HeLa cells and its release from the Au@PEG–CD NPs carrier was detected by a strong increment in its fluorescence intensity. Supporting information available Chemical structure of the monomers, NMR polymer spectrum, additional fluorescence spectra, and Instruments and Experimental Details are shown in the Supporting Information. Acknowledgements G.M would like to thank the Universidad de Alcalá for her postdoctoral contract. Authors are grateful to Isabel Trabado for her support with cell growth and imaging experiments. We wish to express our thanks to M.L. Heijnen for assistance with the preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.01.041. References
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Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry Gema Marcelo ∗ , Ekrem Kaplan 1 , M. Pilar Tarazona, Francisco Mendicuti ∗ Departamento de Química Analítica, Química Física e Ingeniería Química, Universidad de Alcalá, Alcalá de Henares 28871, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 29 November 2014 Received in revised form 21 January 2015 Accepted 23 January 2015 Available online xxx Keywords: Catechol Cyclodextrin Gold nanoparticles Doxorubicin Drug release Polymer
a b s t r a c t A copolymer containing -cyclodextrin, catechol and polyethylene glycol groups in its side chain was designed for the in situ synthesis and coating of gold nanoparticles (Au@PEG–CD NPs). These platforms were designed as a smart carrier and traceable delivery probe of the chemotherapeutic Doxorubicin drug (Dox). The coated polymer forms stable complexes with Dox in water with a high binding constant (K = 2.3 × 104 M−1 at 25 ◦ C), which is one hundred times greater than those reported for its complexation with native CD. Therefore, Au@PEG–CD NPs were able to load 0.01 mg of the drug per mg of NP and to release up to 60% of it in 48 h at 37 ◦ C. In addition, Au@PEG–CD NPs had the capacity to act as a quencher of Dox fluorescence when it was complexed with CD in the NP organic shell. This feature allows the Dox release to be tracked by monitoring the recovery of its fluorescence in real time. Therefore, the Dox release kinetics and the influence of temperature on the thermal stability of Dox/CD complexes on Au@PEG–CD NP were investigated. The increase in temperature favors the dissociation of the complexes and subsequent Dox release from the NP. The first order rate constant for drug releasing was 1.1 × 10−2 min−1 with a half-life time of 63 min at 37 ◦ C. Finally, the great potential of the carrier/probe double nature of Au@PEG–CD NPs was demonstrated in real time inside HeLa cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Doxorubicin (Dox) is a chemotherapeutic drug used in the treatment of a wide variety of cancer types. As with many other drugs, its low water solubility and poor photostability are drawbacks that need to be solved along with the side effects related to its citotoxicity. In order to overcome these limitations Dox has been introduced in different carriers [1–4]. Among these different vectors, gold nanoparticles (Au NPs) are receiving great attention because of their relatively easy surface functionalization [5] and adequate cell penetration [6]. Moreover, Au NPs are known for their optical properties derived from the localized surface plasmon resonance (LSPR), which have a strong impact on a wide range of bioapplications such as imaging, sensing and photothermal therapy [7–9]. An interesting feature is related to the effects that Au NPs induce on organic dye fluorescence properties [10–12]. Au NPs have mostly been reported to act as a dye fluorescence quencher. However, this ability is strongly dependent on the Au NP-dye distance, among other
∗ Corresponding authors. Tel.: +34 918854672. E-mail addresses: [email protected] (G. Marcelo), [email protected] (F. Mendicuti). 1 Current address: Istanbul Technical University, Science and Letters Faculty, Chemistry Department, 34469 Maslak, Istanbul, Turkey.
factors [12]. This behavior renders Au NPs great potential to be used as probes in the monitoring of different biomedical processes such as drug delivery and the determination of key molecules associated to important diseases. For example, Wang et al. [13] and El-Sayed et al. [14] described how Au NPs that bind Dox with an acid-labile linkage, allow the release of Dox into the cell acid lysosomes to be tracked by a recovery of Dox fluorescence after the breaking of the linkage. In another work, Lee et al. used Au NPs functionalized with Cy5.5 to monitor the activity of a protease involved in cancer in vivo. Upon interaction with the protease, the linkage between Cy5.5 and the Au NP breaks off, leading to the recovering of the Cy5.5 fluorescence [15]. On the other hand, cyclodextrins (CDs) are natural cyclic oligosaccharides formed of glucopyranose units. They possess a basket-shaped topology with an inner cavity which exhibits relatively hydrophobic behavior. CDs are able to form reversible, non-covalent inclusion complexes in aqueous media with a wide variety of hydrophobic guests with dimensions that fit inside their cavities. Complexation favors guest solubilization and stability allowing CDs to be used as drug carrier/delivery systems [16]. In addition, complexation processes are usually accompanied by a negative enthalpy change [17], and consequently the amount of the drug released could be increased with temperature, as well as the rate of the process. Another advantage of using CDs as
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carriers is the fact that the transported drug does not need to be chemically modified. Some works dealing with the modification of gold nanoparticles with CDs to transport hydrophobic drugs have been reported [5,18–20], but none of them explores their capability of acting as a probe to obtain information in ‘real time’ about the drug carrier/delivery process. The aim of the present work consisted in designing and preparing surface modified Au NPs with an oligomer which contains -cyclodextrin (CD) in its structure. Subsequently, these hybrid gold–CD NPs (Au@PEG–CD NPs) were investigated as Dox carrier/delivery systems by initially analyzing their cargo capacity and the time required for its release with temperature. The ability of the gold core to act as a quencher of Dox fluorescence was exploited to obtain information about the stability of the drug:CD on gold NP surface complexes, as well as its rate kinetics of release. In addition, the Dox–plasmon interaction allowed us to follow the Dox carrier and subsequent delivery inside the HeLa cells by fluorescence imaging. 2. Materials and methods 2.1. Materials -Cyclodextrin (-CD) (≥98%), p-toluenesulfonyl chloride (99%), sodium azide (99.5%), calcium hydride (CaH2 , 97%), triphenylphosphine (99%), pyridine (99%, ACS reagent), acetone (99.5%, ACS reagent), N,N-dimethylformamide (DMF) (99.8%, ACS reagent), dimethyl sulfoxide (DMSO), borax (Na2 B4 O7 ·10H2 O), magnesium sulfate (MgSO4 ), 2,2 -azobisisobutyronitrile (AIBN), poly(ethylene glycol) methyl ether methacrylate (Mn = 300), methacryloyl chloride, Doxorubicin hydrochloride (Dox) (98.0–102.0%), triethylamine (TEA), chlorhydric acid (HCl) and sodium hydroxide (NaOH) were all purchased from Sigma–Aldrich and used as received. Ethyl acetate (EtAc) was purchased from Scharlau. Hydrogen tetrachloroaurate (III) (HAuCl4 ) and dopamine hydrochloride were obtained from Alfa Aesar. A 0.01 M phosphate buffered saline solution (PBS) of pH = 7.4 (NaCl 0.138 M; KCl – 0.0027 M) (Sigma–Aldrich) was used as a buffer.
2.5. Synthesis of the PEG–catechol–CD polymer A -cyclodextrin monomer (0.5 g), a catechol monomer (0.24 g) and a poly(ethylene glycol) methyl ether methacrylate (2.0 g) were dissolved in 8 mL of oxygen-free DMSO and heated at 83 ◦ C. Azobisisobutyronitrile (AIBN; 6.5 mg) was added in nitrogen atmosphere and the reaction mixture was stirred at 83 ◦ C for 24 h. After that, 50 mL of water (pH = 6) were added to the reaction mixture while stirring. The solution was centrifuged and the supernatant was dialyzed (pore size: 2000 MWCO) for five days. After that, the water was evaporated and the molecular weight of the resulting polymer was characterized by 1 H NMR and UV–vis spectroscopy. The 1 H NMR spectrum of the PEG–catechol–CD oligomer in DMSO is shown in Figure 2S (Supporting Information). 1 H NMR (300 MHz, DMSO-d6), ı (ppm): 6.67 (d, 1H, Aryl-H), 6.64 (s, 1H Aryl-H), 6.53 (d, 1H, Aryl-H), 3.34, 5.78–5.63 (m, 14H in CD), 4.90–4.85 (m, 7H in CD), 4.50–4.45 (m, 6H in CD), 3.66–3.54 (m, 28H in CD), 3.42–3.24 (overlap with HDO, m, 16H in CD), 4.2–3.8 (s, 3H final methyl group in polyethylene glycol chain) and 1.0–0.6 (CH2 and CH of polymer backbone). The mole proportion of each unit in the final structure of the oligomer was calculated taking into account the integral areas of the following signals: the signals between 6.3 and 6.8 ppm for 3H of the catechol aromatic ring, the signal between 6.5 and 6.0 ppm which was assigned to 14H belonging to the CD and the signal between 3.8 and 4.3 ppm corresponding to 3H of the end methyl group in the polyethylene glycol chain. Due to the interaction of the polymer with the columns, the use of the GPC technique to determine the molecular weights was unsuccessful. Therefore, the polymer molecular weight was estimated by UV–vis spectroscopy [24]. First, for a water solution of polymer of well-known concentration in g/L, the molar concentration of the polymer was calculated by using the Beer–Lambert law by using the molar absorptivity of dopamine hydrochloride (2560 M−1 cm−1 in water at 280 nm). Then by combining the concentration (g/L) and the molar concentration (M) it was possible to estimate the molecular weight.
2.2. Synthesis of a methacrylate monomer functionalized with a catechol group
2.6. Determination of the association constants for the Dox with PEG–catechol–CD polymer complexation
The synthesis and NMR characterization of a methacrylate monomer functionalized with a catechol group were performed according to what was described elsewhere [21].
For the 1:1 stoichiometry of the Dox:PolCD (where PolCD means PEG–catechol–CD polymer) complexation process which is described by the following equilibrium,
2.3. Synthesis of mono-6-amino-6-deoxy-cyclodextrin (NH2 -CD)
Dox + PolCD Dox : PolCD
(1)
the association constant K is written as, The synthesis and NMR characterization were carried out as described elsewhere [22]. 2.4. Synthesis of a methacrylate monomer functionalized with ˇ-CD The synthesis and NMR characterization of a methacrylate monomer were performed according to what was reported [23].
I = I0 + (I∞ − I0 )
(1 + K[Dox]0 + K[PolCD]0 ) −
K=
[Dox : PolCD] [Dox][PolCD]
(2)
where [PolCD], [Dox] and [Dox:PolCD] are the concentrations of the PEG–catechol–CD (per mol of CD), Dox and the 1:1 complex respectively. By assuming two fluorescent species at the equilibrium, the free Dox and the complexed one, Dox:PolCD, the binding constants can be determined from the non-linear dependence on fluorescence intensity with the total initial PEG–catechol–CD concentration, [PolCD]0 , according to the following expression:
2 (1 + K[Dox]0 + K[PolCD]0 ) − 4K 2 [Dox]0 [PolCD]0 2K[Dox]0
(3)
where I, I0 and I∞ are the corrected fluorescence intensity at each [PolCD]0 , for the free Dox (in the absence of a PEG–catechol–CD) and for the complex (at [PolCD]0 →∞) respectively. [Dox]0 and [PolCD]0 are the initial concentrations of the Please cite this article in press as: G. Marcelo, et al., Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.01.041
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drug guest (=5 × 10−7 M) and the PEG–catechol–CD derivative. Eq. (3) assumes that the Dox fluorescence intensity is quenched upon PEG–catechol–CD addition. Protocols: fluorescence spectra for a set of solutions, named A, of PEG–catechol–CDs with concentrations increasing up to 14 mg/mL in the presence of a constant concentration of Dox (5 × 10−7 M) in a PBS buffer, and for another set, named B, with exactly the same PEG–catechol–CD concentrations but in the absence of Dox, were recorded. In order to remove the strong scattering on emission due to polymer aggregation with the concentration, each emission spectrum was corrected by subtracting at each spectrum of set A the corresponding one of set B. The Dox/PEG–catechol–CD PBS solution emission spectra after subtraction are shown in Figure 3S (Supporting Information). 2.7. In situ formation and surface modification of gold nanoparticles with PEG–catechol–CD 150 mg of PEG–catechol–CD were dissolved in 17 mL of MiliQ water. Then 1 mL of HAuCl4 water solution (16 mg/mL) was added under magnetic stirring (300 rpm). Direct observation of the Au NP formation took place after 3 min, the solution became blue. The mixtures were left stirring for 20 min. The resulting particles were cleaned by redispersion in distilled water by the use of an ultrasound bath (Elma). After that, they were stirred for 10 min and finally they were centrifuged. This process was repeated three times. 2.8. Determination of amount of Dox adsorbed on Au@PEG–CD NPs Dry Au@PEG–CD NPs were re-dispersed in a Dox PBS solution (2.5 × 10−5 M). After that, the colloidal dispersion was left stirring overnight. The amount of Dox loaded in the Au@PEG–CD NPs was obtained by UV–vis spectroscopy. To do this, the absorbance of the initial Dox solution without gold nanoparticles and the absorbance of the final supernatant solution after isolating the gold nanoparticle by centrifugation (12,000 rpm) were determined. The extinction coefficient was 11,500 cm−1 M−1 [25]. 2.9. The kinetics of Dox release Two samples of Au@PEG–CD NPs (8 mg) were re-dispersed in either 23 mL of Dox 1.0 × 10−6 M (in PBS) and 4.3 mL of Dox 2.2 × 10−5 M (in PBS). Colloid dispersions were left stirring overnight. After that, NPs were isolated by centrifugation. The amount of Dox adsorbed on NPs was determined by UV–vis spectroscopy. The Dox concentration in each one was determined to be 1.0 × 10−6 M and 1.1 × 10−5 M. Then, they were re-dispersed in 4.3 mL of fresh PBS to obtain two stock colloidal dispersions which were stored at 4 ◦ C. To study the kinetics of Dox release, an aliquot of both stock colloidal dispersions was re-dispersed in a fresh PBS buffer. Thus, the final Dox concentrations in the colloidal dispersion were 3.3 × 10−8 M and 3.6 × 10−7 M (0.06 mg/mL of Au@PEG–CD NPs in PBS) and the steady-state fluorescence spectrum was tracked with time. The experimental time depended on the temperature of the experiments. Spectra were collected until the fluorescence intensity remained constant. The experimental times were 510, 450 and 410 min at 25, 37 and 45 ◦ C respectively. 2.10. Cell growth and interaction with the [Au@PEG–CD NPs. . .Dox] complexes HeLa cells (ATCC CCL-2) were kindly served by the cells culture unit of UAH. Cells were cultured with an RPMI1640 medium containing 10% fetal bovine serum (FBS, Sigma Ref. F7524) and
3
10% antibiotic antimycotic solution (Sigma, Ref. A5955) at 37 ◦ C under 5% CO2 , 95% air-humidified atmosphere. The culture media was changed every 2 days. After that, HeLa cells were seeded into 60 -Dish 35 mm glass bottom (Ibidi, Ref. 81158) at an initial cell density of 2 × 104 cells/dish. After 48 h, 50 L of PBS containing Au@PEG–CD NPs (2 mg/mL) complexed with Dox at two different concentrations, 2.8 × 10−6 and 8.2 × 10−5 M, were added and the cells were incubated. Cells were imaged at different incubation times depending on the system.
3. Results and discussion Throughout the different sections of this manuscript, we will initially explain the design, synthesis and characterization of the polymer containing CDs which will be used to both prepare and modify the Au NPs surface. In addition, the thermodynamics of the complexation process in aqueous medium between this polymer and Dox will be studied. Subsequently the strategy for both the preparation and the modification of Au NPs with the polymer will be described and the carrier capability, its interaction with Dox and the kinetics of the drug release will be investigated. In the last part, the drug-carrier interaction will be analyzed in a more realistic system after its uptake in HeLa cells.
3.1. Preparation and characterization of a cyclodextrin polymer (PEG–catechol–CD) A (PEG–catechol–CD) copolymer was synthesized by the radical polymerization reaction of poly(ethylene glycol) methyl ether methacrylate and two methacrylamide derivatives containing cyclodextrin (CD) and catechol moieties respectively (structures of the monomers are depicted in Figure S1 of the Supporting Information). Each monomer plays an important role in the function of the final colloid system: the CD macroring from the first monomer provides an effective site for the drug interaction, the PEG gives both hydrophilic behavior and biocompatibility [26] to the system and the catechol groups from the third monomer are reducing agents versus HAuCl4 allowing for the formation of Au NPs [27,28]. Scheme 1 shows the chemical structure of the PEG–catechol–CD copolymer. 1 HNMR together with UV–vis spectroscopies allowed us to characterize the copolymer. The composition of the side chain was: 6.5 mol % CD, 14.7 mol % catechol groups and 78.8 mol % of poly(ethylene glycol) chains. The molecular weight was estimated to be 6000 g mol−1 . Therefore, each oligomer is composed on average of 12 units containing poly(ethylene glycol), two units bearing catechol groups and one with a pendant CD macrocycle. The water behavior of the PEG–catechol–CD copolymer was analyzed by DLS. The variation of the hydrodynamic diameter with the polymer concentration at 25 ◦ C is depicted in Fig. 1. The PEG–catechol–CD copolymer exhibits a strong capacity to form aggregates above a 7 g/L concentration. Its hydrodynamic diameter is greater with the increase of polymer concentration, reaching 120 nm for a polymer concentration of 100 g/L. We explained the aggregation behavior with the increase in polymer concentration considering the moderate hydrophobicity of catechol groups [29]. We consider that catechol groups buried inside aggregates via hydrophobic and – stacking interactions form an inner core, while the highly hydrophilic PEG chains, as an outer shell layer, surround it. FE-SEM characterization for a PEG–catechol–CD water solution (16 mg/mL) shows spherical aggregates of polymer with a diameter ranging between 100 and 150 nm. This high value in relation to the hydrodynamic diameter could be due to an aggregation process during the water evaporation on the carbon grid used for
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Scheme 1. Chemical structure of PEG–catechol–CD copolymer and its schematic representation.
Fig. 1. (A) Hydrodynamic diameter as a function of polymer concentration. (B) FE-SEM of a dried drop of polymer solution (16 mg/mL).
the SEM characterization. Nevertheless, it could be indicative of the presence of self-assembling polymer structures.
3.2. Interaction of the PEG–catechol–CD with Dox The complexation process of Dox with the PEG–catechol–CD polymer illustrated in Fig. 2 (top) was studied in a PBS buffer solution by using fluorescence spectroscopy. The Dox concentration was fixed at 5 × 10−7 M and the PEG–catechol–CD concentration was increased up to 14 g/mL. Dox concentration was sufficiently low to avoid dimer formation. The complexation of Dox with the PEG–catechol–CD is characterized by some changes in the photophysical properties of Dox. A decrease in its corrected fluorescence intensity and an increase in its weighted average fluorescence lifetimes, , obtained from fluorescence decay profiles (equation 2S supporting information) were observed. Changes are attributed to the Dox inclusion into CD cavities. Some preliminary experiments on Dox in the presence of a PEG–catechol polymer which lack CDs, exhibit not changes in the fluorescence intensity with concentration. The changes of both properties for Dox at different PEG–catechol–CD concentrations are depicted in Fig. 2(A) and (B) respectively. The variation of the fluorescence intensity with polymer concentration allows us to determine the binding constant for the Dox complexation with PEG–catechol–CD. The curve depicted in Fig. 2(A), obtained by adjusting the experimental data from steadystate measurements to the proper equation for a 1:1 Dox:CD
Fig. 2. Top: Illustration of the complexation process between the PEG–catechol–CD and Dox. (A) Corrected fluorescence intensity measured at 590 nm (exc = 480 nm) and (B) weighted average lifetime for Dox at [Dox]0 = 5 × 10−7 M and different concentrations of PEG–catechol–CD (PolCD) (expressed in CD molar concentration) measured at 600 nm upon excitation wavelength of 360 nm.
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Fig. 3. Top: Illustration of the redox reaction that leads to the in situ formation and functionalization of gold nanoparticles with a coating of PEGMA–catechol–CD. (A) FE-SEM image of Au@PEG–CD NPs. (B) UV–vis–NIR absorption spectrum of Au@PEG–CD NPs.
stoichiometry (Eq. (3)) shows a reasonable fit. This provided a binding constant of (2.3 ± 1.7) × 104 M−1 at 25 ◦ C. The curve depicted for the variation in Fig. 2(B) was also obtained by fitting the experimental data to an equation similar to 3 (see Supporting Information, equation 3S), but using instead of the fluorescence intensity and the K value obtained from steady-state fluorescence intensity measurements. The value binding constant of 2.3 ×104 M−1 is two magnitude orders higher than the one obtained for the Dox complex with native CD at the same temperature. The incorporation of CDs in a polymeric structure leads to a cooperative inclusion behavior enhancing the binding affinity of CDs with a broad spectrum of drugs [30,31]. For instance, a binding constant of 3.7 × 104 M−1 was reported between the chemotherapeutic drug albendazole and a block copolymer modified with CDs. This value was 23 times higher than that obtained for the albendazole complexation with the native CD [31]. According to this result, the PEG–catechol–CD could be considered an excellent system to modify the Au NPs in order to obtain a stable carrier for Dox.
were determined in water by UV–vis–NIR spectroscopy, exhibiting the LSPR [32] located at 650 nm. Moreover, two more bands centered at 285 and 400 nm, as Fig. 3(B) depicts, were also observed. The higher energy band corresponds to the catechol groups and the other one was attributed to a catechol oxidation product formed during the redox reaction [33–35]. To complete the characterization, thermo gravimetric analysis allowed us to determine the weight percent of the organic coating in the Au@PEG–CD NPs that was 25 wt %. Therefore, there was 0.25 mg of polymer in 1 mg of Au@PEG–CD NPs. The number of CD moles was estimated to be 4.7 × 10−8 per mg of Au@PEG–CD NPs. 3.4. Interaction of Au@PEG–CD NPs with Dox and the kinetics of Dox release The potential of Au@PEG–CD NPs to act as a Dox carrier was evaluated in terms of their drug loading capability and
3.3. Preparation of gold nanoparticles and functionalization with a PEG–catechol–CD coating It has been reported that catechol groups suffer an oxidative self-conversion into their quinone forms by releasing protons and electrons under mild reductive conditions [27,28]. They can then be used for the gold salt precursor reduction to form Au NPs. Therefore, the Au NPs modified with PEG–catechol–CD copolymer were prepared in situ by stirring during 20 min a water solution of PEG–catechol–CD copolymer in the presence of an excess of HAuCl4 . After 4 min, the solution gradually turned blue. The resulting particles were named Au@PEG–CD NPs. See the scheme in Fig. 3 (Top). FE-SEM of Au@PEG–CD NPs of Fig. 3(A) showed gold nanoparticles with a size of ca. 90 nm. A thick shell of PEG–catechol–CD polymer covers the Au NP. Optical properties for Au@PEG–CD NPs
Fig. 4. Quantification of the release of Dox from Au@PEG–CD NPs in a PBS buffer solution with time at two temperatures: 25 ◦ C () and 37 ◦ C ( ).
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Fig. 5. Top: Illustration of the influence of the temperature in both the Dox delivery and trigger ON of its fluorescence. (A) Raw fluorescence emission spectra with time of Au@PEG–CD NPs loaded with Dox (7 × 10−7 M) in PBS at 25 ◦ C, (exc = 480 nm). (B) Fitting of experimental fluorescence data to a first-order kinetic reaction at 25 (), 37 ( ) and 45 ◦ C ( ).
subsequent release with time feasibility. The estimated maximum amount of Dox complexed with the Au@PEG–CD NPs (Au@PEG–CD NPs. . .Dox) was 0.01 mg per mg of NPs (1.7 × 10−8 moles per mg of NPs). According to the CD mole content determined above, this value indicates that Dox is adsorbed on Au@PEG–CD NPs in nearly an equimolar concentration in relation to CD. The amount of Dox released at different times was determined at two temperatures, 25 and 37 ◦ C. We assume that the re-dispersion of (Au@PEG–CD NPs. . .Dox) in a fresh PBS buffer leads to a de-complexation process, in which the maximum Dox amount freed corresponds to its equilibrium concentration. Therefore, at equilibrium, Dox can either appear as free Dox or as complexed (Au@PEG–CD NPs. . .Dox) forms. Fig. 4 shows the percentage of drug released with time which, as expected, depends strongly on the temperature. After 48 h, this amount reached 23% at 25 ◦ C, but at 37 ◦ C it was 60%. This result can be explained considering that the Au@PEG–CD NPs. . .Dox complexation process was governed enthalpically. As a result, the complex stability constant diminishes with temperature [19,20].
As stated earlier in this paper, when a chromophore molecule close to the gold NP surface interacts with the gold NP plasmon field, it results in a quenching of its fluorescence [15,36]. Therefore, the recovery of fluorescence upon Dox release from Au@PEG–CD NPs can be a useful tool to obtain information in real time about the thermal stability of the Dox. . .CD complexes on the carrier and to follow the kinetics of drug release in solution. Steady-state fluorescence intensity was used to monitor the release with time of Dox from Au@PEG–CD NPs for two colloidal solutions of (Au@PEG–CD NPs. . .Dox) in PBS containing Dox at two different concentrations 3.3 × 10−8 M and 3.6 × 10−7 M at three temperatures: 25, 37 and 45 ◦ C. For the colloidal solution with Dox at 3.3 × 10−8 M, Dox fluorescence was initially not detected and even with the recorded time no emission was observed for any of the temperatures. This might be because of either the very low Dox concentration or the overlapping of its emission with the NPs dispersion scattering band. However, an initial emission of Dox was observed together with the background NPs scattering for the colloidal solution containing Dox at a concentration of 3.6 × 10−7 M.
Fig. 6. Images of fluorescence (in red) obtained with Laser scanning confocal microscopy for the [Au@PEG–CD NPs. . .Dox] dispersion in a PBS buffer at 37 ◦ C after 30 min (left) and 180 min (right). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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The variation of the emission band with time at the three temperatures was studied. As an example, Fig. 5(A) depicts the emission spectra changes at 25 ◦ C. There is an increment in intensity of the Dox emission band with time at any temperature, which is attributed to the increase in the amount of fluorescent free Dox. Releasing kinetics was quantified at these temperatures by adjusting the experimental data to first-order reactions. Rate constant values were 7.9 × 10−3 , 1.1 × 10−2 and 2.2 × 10−2 min−1 with half-life times (time in which the 50% of Dox is released) of 88, 63 and 32 min at 26, 37 and 45 ◦ C respectively. The decomplexation process is therefore thermally favored and the delivery rate increases with temperature. Evident information of the Dox release at 37 ◦ C was obtained by laser scanning confocal microscopy upon monitoring the images of a dispersion of (Au@PEG–CD NPs. . .Dox) in a PBS buffer (2 mg/mL of Au@PEG–CD NPs with a concentration of Dox of 2 × 10−6 M) at two different times, 30 min and 180 min. As Fig. 6 shows, a significant enhancement of Dox fluorescence emission intensity after 180 min was observed, which is due to the release from Au@PEG–CD NPs. 3.5. Interaction of (Au@PEG–CD NPs. . .Dox) with HeLa cells Most of the time, drug delivery from the carrier occurs with hardly noticeable changes of the fluorescent properties neither in the carrier nor the drug [37]. Only very few studies, where the drug release leads to changes in the drug fluorescence intensities and lifetimes, were reported [3,13,14]. In this next section, we demonstrate that Au@PEG–CD NPs can act simultaneously as an effective cellular carrier and as a probe to track in real time the Dox cellular release in a living system by monitoring the increment in its fluorescence intensity. HeLa cells were incubated at 37 ◦ C with (Au@PEG–CD NPs. . .Dox) containing Dox at two different, 7 × 10−8 and 2 × 10−6 M, concentrations. These concentrations were not very different from those used in the previous steady-state fluorescence experiments of the preceding section. The cellular uptake of (Au@PEG–CD NPs. . .Dox) at 30 min and 240 min of incubation time was investigated by recording CLSM images. For a Dox concentration of 7 × 10−8 M there is no Dox fluorescence emission after 30 min of incubation (data not shown). Dox is hardly released from Au@PEG–CD NPs at this time and its emission is totally quenched. However, as depicted in Fig. 7, after 240 min of incubation, a weak fluorescence emission of Dox was detected in the cell cytoplasm. Therefore, even at the low concentration used, the experiments enable us to corroborate that the Au NP also acts as a very
Fig. 7. Laser scanning confocal microscopy images of HeLa cells treated with Au@PEG–CD NPs. . .Dox containing a [Dox] = 7 × 10−8 M after 240 min.
sensitive probe to study the drug releasing in cells by turning on its fluorescence upon delivery. For a Dox concentration of 2 × 10−6 M after 30 min of incubation, as depicted in Fig. 8, the Dox fluorescence emission can be perfectly observed in the cytoplasm of HeLa cells. At this Dox concentration, according to the kinetics of release, only 63 min is necessary for a 50% of Dox release at 37 ◦ C. The Dox emission intensity in the inner cells increases considerably after 240 min of incubation, namely after four times the half-life time. This fact could be attributed mainly to a greater amount of Dox released from the carrier together with a greater internalization of the (Au@PEG–CD NPs. . .Dox) complexes. HeLa cells incubated with only the Dox drug at 6.0 × 10−7 M presented a high degree of internalization (see Supporting Information, Figure S4). The fluorescence of Dox was observed in the cytoplasm as well in the nuclei at 240 min of incubation. Free Dox is known to be transported into cells via diffusion [4]. The difference in the localization and internalization degree of free Dox compared with when it is transported as (Au@PEG–CD NPs. . .Dox) could be attributed to the different cellular uptake mechanisms.
Fig. 8. Laser scanning confocal microscopy images of HeLa cells treated with Au@PEG–CD NPs. . .Dox containing a [Dox] = 2 × 10−6 M after different incubation times: (A) 30 min and (B) 240 min. Scale bar 50 m.
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4. Conclusions Au NPs modified with a polymeric shell containing CDs were prepared, Au@PEG–CD NPs. Their capability to act simultaneously as both a Dox carrier and a probe to monitor the Dox–CD interaction on the carrier was demonstrated in solution and in a living system as well. The preparation of these gold nanoparticles was carried out in two steps: (i) synthesis of a copolymer which contains catechol, CD and PEG groups that present a remarkable high binding affinity toward Dox (K = 2.3 × 104 M−1 at 25 ◦ C); and (ii) in situ formation and decoration of gold nanoparticles with the copolymer by using the catechol redox chemistry. 1 mg of Au@PEG–CD NPs was able to complex 0.01 mg of Dox and to release it in a 60% after 48 h at 37 ◦ C. The interaction between Dox and the LSPR led to the quenching of Dox fluorescence. Therefore, by analyzing the recovery of Dox fluorescence after its delivery, quantitative information about the Dox release kinetics was obtained. An increase in the temperature contributed notably to the dissociation of the Au@PEG–CD NPs. . .Dox complex and subsequent drug release. To conclude, the success of the double nature carrier/probe was demonstrated in a living system in real time. Dox is transported to the cytoplasm of HeLa cells and its release from the Au@PEG–CD NPs carrier was detected by a strong increment in its fluorescence intensity. Supporting information available Chemical structure of the monomers, NMR polymer spectrum, additional fluorescence spectra, and Instruments and Experimental Details are shown in the Supporting Information. Acknowledgements G.M would like to thank the Universidad de Alcalá for her postdoctoral contract. Authors are grateful to Isabel Trabado for her support with cell growth and imaging experiments. We wish to express our thanks to M.L. Heijnen for assistance with the preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.01.041. References
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