Piersandro
Pallavicini
*a,
Valeria
Amendola
*a,
Greta
Bergamaschi
a,
Elisa
Cabrini
a,
Giacomo
Dacarro
ab,
Nadia
Rossi
a and
Angelo
Taglietti
a
aDipartimento di Chimica, Università degli Studi di Pavia, v.le Taramelli 12, 27100 Pavia, Italy. E-mail: piersandro.pallavicini@unipv.it; valeria.amendola@unipv.it; Fax: +39 0382528544; Tel: +39 0382987336 Tel: +39 0382987329
bDipartimento di Fisica, Università degli Studi di Pavia, via Bassi 6, 27100 Pavia, Italy
First published on 25th January 2016
A new macrobicyclic ligand capable of binding two Cu2+ cations has been synthesized and its protonation and coordinative properties fully determined in aqueous solution. A thioether moiety was appended on the ligand backbone. This does not influence the ligand coordination ability but allows us to graft its bis-copper complex on the surface of a self-assembled monolayer of gold nanostars (GNS), in turn grafted on glass slides pre-functionalized with a layer of a silane-bearing polyethyleneimine polymer. The release of copper ions from the GNS monolayers was also investigated, finding a general agreement with the coordination properties of the complex in solution, although the bis-copper complex displays an increased kinetic inertness when grafted on the glass slides. The photothermal properties of the GNS monolayer were studied with and without the overlayer of the Cu2+ complex, finding no influence of the latter but disclosing that the bis-copper complex detachment is promoted by local T increase due to laser irradiation.
Gold nanostars (GNS) are gold nanoparticles with 2–6 branches protruding from a core and featuring the peculiar optical properties of non-spherical gold nanoparticles.6 In particular, we recently prepared GNS using the Triton X-100 surfactant in a seed-growth approach.7 Such GNS are characterized by two localized surface plasmon bands (LSPR) in the near IR (NIR) region of the spectrum. Both of these LSPR display an intense photothermal effect, i.e. once excited with a laser radiation they relax thermally, allowing the conversion of radiation into heat with a local T jump, and offering two photothermally active channels in a spectral region (NIR) in which blood and tissues are transparent.7,8 The photothermal effect exerted by monolayers of GNS anchored on a surface can be exploited to obtain an antibacterial and antibiofilm action. This is triggered by through-tissues laser irradiation and opens a new approach in the surface modification of internalized medical devices (e.g. prostheses, implants, catheters).9
In addition, GNS prepared with seed-growth synthesis with TritonX-1007 or lauryl sulfobetaine (LSB),10 due to the weak surfactant–surface interactions can be functionalized on their surface with thiols through straight-forward mix-and-obtain procedures,7,10,11 differently from what observed e.g. with gold nanorods obtained with seed-growth syntheses using strongly bonding cationic surfactants.12 We recently exploited such property to obtain the first example of GNS co-coated with a Cu(II) complex and a stabilizing thiol (i.e. a thiol-terminated polyethyleneglycol)13 and to prepare GNS co-coated with a thiolated derivative of the bodipy dye and with a thiol-terminated polyethyleneglycol.14 In the latter case we also demonstrated that the thiol–gold bond can be weakened by T increase, so that release of a thiol-grafted molecule could be triggered also by laser irradiation, exploiting the photothermal effect.
In this paper we present GNS monolayers on glass, overcoated with the Cu(II) complex of a new bistren cryptand (L, Scheme 1), featuring a thiolated arm on the backbone, suitable for grafting on the GNS surface. A layer-by-layer approach was used, first coating glass slides with a siloxane derivative of the PEI (polyethyleneimine) polymer, PEI-s, obtaining Type I surfaces in Scheme 1. On these, we grafted GNS prepared with TritonX-1007 (Type II surface, Scheme 1), that were further overcoated with Cu2+ complexes of the L ligand, [Cu2L]4+, to obtain Type III surfaces. We investigated different strategies for the final step of functionalization, in order to obtain the best loading of the complex on Type II surfaces. Although papers have been published on colloidal solutions of metal nanoparticles bearing metal complexes on their surface,15 to the best of our knowledge this is the first example of a layer-by-layer functionalization of bulk surfaces involving metal nanoparticles and transition metal complexes.
Scheme 1 Formula and schematic representation of the molecular species and surface types mentioned in this paper. |
The protonation and coordination properties of L towards Cu2+ have been examined in solution by means of potentiometric titrations, and compared with the stability of the complexes as monolayers grafted on the GNS monolayer in contact with aqueous solutions, at given pH values. Moreover, the photothermal properties of the Type II and Type III surfaces have been studied. We demonstrated that the overlayer of [Cu2L]4+ does not affect the radiation to heat conversion and that the complex release can be promoted by the local T jump induced by laser irradiation.
Protonated speciesa | LogK valuesb | Complex speciesc | LogK valuesb |
---|---|---|---|
a Protonation constant of the species LHn refers to the L + nH+ = [LHn]n+ equilibrium. b Uncertainties in parentheses. c Formation constants refers to equilibria L + nH+ + mCu2+ = [CumHn](2m+n)+ (m = 1, 2; n = 0, 2, 3) The formation constant of species LCu2OH refers to the equilibrium L + 2Cu2+ + H2O = [LCu2(OH)]3+ + H+. | |||
LH1 | 9.2(1) | LH3Cu | 31.9(1) |
LH2 | 17.6(1) | LH2Cu | 27.2(2) |
LH3 | 25.2(1) | LCu2 | 19.0(2) |
LH4 | 31.8(1) | LCu2OH | 11.8(1) |
LH5 | 37.9(1) |
In the pH-spectrophotometric titration experiment the UV-Vis absorption spectrum of the solution was recorded after each addition of standard 0.1 M NaOH.
MS (ESI, MeOH, pos.): m/z 345 [M + 2H]2+, 689 [M + H]+.
1H NMR (400 MHz, CD3OD, ppm): δ 2.13 (3H, s, Ha), 2.65 (12H, t br, Hj, Hj′), 2.8 (14H, m br, Hk, Hk′, Hb), 3.7 (8H, m, Hi, Hi′), 4.15 (2H, t, Hc), 6.4 (1H, d, He), 6.9 (4H, d, Hh), 7.0 (5H, m, Hf, Hg), 7.3 (1H, s, Hd).
13C NMR (400 MHz, CD3OD ppm): δ 13.5, 32.0, 52.0, 52.5, 52.7, 66.1, 110.0, 118.6, 125.5, 126.5–127.0, 137.0, 138.2, 139.3, 155.0.
Among the possible TritonX-100 and ascorbic acid concentrations (leading to different aspect ratios in the branches) for this paper we used the following conditions: a seed solution was prepared in a 20 mL vial, in which 5 mL of HAuCl4 5 × 10−4 M in water were added to 5 mL of an aqueous solution of Triton X-100 0.2 M. The mixture was gently hand-shaken taking a pale yellow colour. Then, 0.6 mL of an ice-cooled solution of NaBH4 0.01 M in water were added. The mixture was gently hand-shaken and a reddish-brown colour appeared. The seed solution was kept in ice and used within 2 hours. The growth solution was prepared in a 250 mL Erlenmeyer flask. 2.50 mL of AgNO3 0.004 M in water and 50 mL of HAuCl4 0.001 M in water were added in this order to 50 mL of an aqueous solution of TritonX-100 0.2 M. Then, 1400 μL of an aqueous solution of ascorbic acid 0.0788 M were added. The solution, after gentle mixing, became colourless. After this, 120 μL of the seed solution were added. The solution was gently hand-shaken and a grey colour appeared and quickly changing to more intense and blue-black colors. The sample was allowed to equilibrate for 1 h at room temperature before using it for the coating procedures (see ESI,† Fig. S2 for an extinction spectrum). ICP-OES analysis (on ultracentrifuged pellets oxidized with aqua regia) allowed to determine the concentration of Au (∼60 μg mL−1, corresponding to 61% yield). Under these synthetic conditions, the extinction spectrum of such GNS presents two LSPR bands, centered at 850 and 1630 nm.
Only five protonation constants were observed in the explored pH range, relative of five of the six secondary amines of the aza-cryptand L (the more hindered tertiary amines do not protonate under such conditions20,22). An analogous titration has been carried out in the presence of 2 equivalents of the Cu(II) salt Cu(CF3SO3)2. The best data fitting was obtained by considering the formation of the following copper species on increasing pH: [CuLH3]5+, [CuLH2]4+, [Cu2L]4+ and [Cu2L(OH)]3+. The formation constants of such species are listed in Table 1. From the protonation and formation constants, we can calculate and draw the distribution diagram of the species in solution (% abundance with respect to total Lvs. pH). This is shown in Fig. 1A. At pH 2.5, the azacryptand is in the pentaprotonated form, [LH5]5+ (100%). Upon addition of NaOH, the monometallic complex [CuLH3]5+ forms and reaches its maximum concentration (40%) at pH 4.7 (see Fig. 1A).
We may hypothesize that in [CuLH3]5+ the three protons are on a single tren subunit, while the second one is occupied by the Cu2+ ion. On increasing the pH, a proton is lost and the mononuclear copper species [CuLH2]4+ forms (maximum abundance 61% at pH = 5.3). The dicopper complex, [Cu2L]4+ is the major species in solution at pH 6.5 (75%). In [Cu2L]4+ each Cu2+ ion occupies one of the tren units, adopting the typical trigonal-bipyramidal geometry imparted by tripodal ligands. The fifth (apical) coordination position of each metal ion is occupied by a water molecule. Upon further addition of NaOH to the complex solution, the deprotonation of the coordinated water occurs, leading to the stable hydroxide complex, [Cu2L(OH)]3+. Such species are typical of bis-copper complexes of bis-tren aza-cryptands, with the OH− ion bridging between the two copper centers.26 Due to its high stability, this species predominates in solution (>90%) over the 8–11 pH range. In addition, it has to be pointed out that from the distribution diagram it can be seen that at pH ≥ 7.0 only dicopper species exist, either as a mixture of [Cu2L]4+ and [Cu2L(OH)]3+ (7.0 < pH < 9.0) or as [Cu2L(OH)]3+ (pH > 9.0). Fig. 1B and C show the family of spectra taken over the course of the pH-spectrophotometric titration of a solution of L (0.8 mM) and Cu(CF3SO3)2 (1.6 mM). The formation of the copper complexes is accompanied by the development of a band around 280 nm, attributable to a LMCT N(amine) → Cu(II), see Fig. 1B. Moreover, two d–d bands develop in the visible region in the 650–850 nm range (see Fig. 1C), typical of bipyramidal Cu(II) complexes.22,26 The profile of Absorbance at 800 nm vs. pH (red triangles in Fig. 1A) fits well with the formation of the [Cu2L]4+ and [Cu2L(OH)]3+ species in the distribution diagram. Noticeably, the spectrum taken at pH 7 (blue lines in Fig. 1B and C, maximum% of [Cu2L]4+) and at pH 11 (green lines in Fig. 1B and C, 100% [Cu2L(OH)]3+) show slight but sharp differences, evidencing the different coordinative sphere of the Cu2+ cations between the two species.
Au concentration in the GNS colloidal solution is 60 μg mL−1 (61% conversion yield from AuCl4−), calculated separating GNS from solution by ultracentrifugation and analysing the obtained pellet by ICP-OES after full oxidation in a given volume of aqua regia. Grafting GNS on PEI causes a ∼50 nm blue shift of the first LSPR (and a ∼300 nm blue shift of the second one), due to the well-known sensitivity of LSPR bands to the local refractive index. The latter changes from 1.3339 (water) in colloidal solution to that of a mixed medium composed mainly of air (1.0003) and of PEI (on which the GNS are adhering). The wavelength maximum of the first LSPR in solution is chosen in order to obtain an absorption maximum at ∼800 nm on Type II surfaces (see Fig. 2B, blue line). This is the wavelength of the laser source available in our laboratories. We used 18 h dipping time to prepare Type II surfaces, to be sure to obtain the maximum surface coating. Gold content was determined by full oxidation of the GNS monolayer with aqua regia and standard analysis by ICP-OES. An average of 4.7(±0.3) μg Au per cm was found on 3 different preparations, in agreement with what observed previously for the same GNS but with lower aspect ratio of the branches (first LSPR at 630 nm, second at 1100 nm when grafted on glass in ref. 7). SEM imaging, Fig. 2C, show a homogeneous distribution of the GNS lying flat on the surface, forming a (sub)monolayer. Due to their irregular shape, close packaging of GNS is obviously not allowed and unoccupied space is left on the surface. Note that the less sharp GNS morphology in the SEM image is due to the coating with ∼5 nm of sputtered graphite, added to ensure the necessary sample conductivity.
Fig. 2 (A) Extinction spectra of Type II surface (blue color) and of Type III surface obtained from it (red color); (B) TEM image of a GNS used for coating (see ESI,† Fig. S3 for complete image); (C) SEM image of a Type II surface (particular; see ESI,† Fig. S1 for full image and additional images). |
Type III surfaces feature an additional overlayer of the bis-Cu2+ complex of cryptand L. To obtain such surfaces we have used a two-step approach (TSA) and a single-step approach (SSA). In the TSA, the empty ligand is first grafted on the free surface of GNS by dipping Type II glass slides in a 10−4 M aqueous solution of L. Slides were then repeatedly washed (see Experimental) to remove the L ligand not grafted on the surface. Finally they were immersed in a 10−4 M solution of Cu2+ (as its triflate salt) for 4 hours, then repeatedly washed with bidistilled water. ESI† (Fig. S4a) reports the extinction spectra of the starting surface (Type II) and of the two steps. With the SSA, the [Cu2L](CF3SO3)2 complex was instead pre-prepared and isolated as described in the Experimental. Type II slides were immersed for 4 h in a 10−4 M solution of such complex, regulated at pH 7 by NaOH microadditions, and then repeatedly washed with bidistilled water. With both approaches the LSPRs of the GNS shifted significantly to the red, indicating a change of the local refractive index around GNS.27 Interestingly, with the TSA we observed first a small red shift when the empty ligand was added, and then an additional, more significant red shift when Cu2+ was added to the modified surfaces, see Table 2 and ESI† (Fig. S4A). In the case of SSA, the observed red shift is instead smaller than that observed after the second step of the TSA (Table 2). Fig. 2A displays the extinction spectrum of Type III surface prepared with SSA (red spectrum), evidencing such red shift. ICP-OES analysis was carried out to determine the total quantity of Cu2+ on the surface after the two approaches. Data are collected in Table 2 and show that the TSA yields surfaces with a significantly lower quantity (63% lower) of Cu2+ with respect to the SSA preparations. A larger Δλ shift is observed in the TSA, particularly in the second step, when Cu2+ is added to the surface with grafted but void ligand L. This observation and the found low Cu2+ surface concentration suggest that the added free Cu2+ cations interact with the GNS environment, but do not enter (or enter just in small percent) inside the surface-confined bis-tren cage. Complexation to the free portions of PEIs can be hypothesized, as glass-grafted PEIs has already been proven to weakly bind Cu2+ from aqueous solutions.21 Experiments carried out immersing the Type II surfaces in 10−4 M Cu(CF3SO3)2 support such hypothesis, as similar shifts of the LSPR bands are observed (ESI,† Fig. S4B). This picture is not surprising, considering the already observed surface kinetic effect, hugely slowing the complexation/decomplexation processes taking place on ligands grafted at a short distance to a bulk surface.28 On the other hand, the SSA employs the preformed [Cu2L](CF3SO3)4 complex and leads to higher Cu2+ surface concentrations. The obtained ∼0.35 nmol Cu2+ per cm concentration corresponds to a ∼0.175 nmol cm−2 concentration of [Cu2L]4+.29 This value is well inscribed inside what found for molecular monolayers of Cu2+ complexes grafted by a pendant function on flat glass. Examples are the Cu2+ complex of a tetraaza macrocyclic ligand (0.22 nmol cm−2) and of the 2,2-bipyridine ligand (0.17 nmol cm−2).28 In the present case the surface offered for grafting [Cu2L]4+ is of course different from a flat glass slide, as it is made of GNS in turn grafted on glass. Such surface has a nanostructured shape capable of increasing the available surface: an higher surface concentration of grafted complex could be expected. On the other hand the GNS monolayer on Type II surfaces is not densely packed, see Fig. 2C, and this lowers the surface concentration of the grafted complex. On the basis of these data, the SSA has been used in all subsequent studies.
Synthetic approach | Δλ (LSPR1)a | Δλ (LSPR2)b | Cu2+ per cm |
---|---|---|---|
a LSPR1 is the LSPR at ∼800 nm for Type II surfaces. b LSPR2 is the LSPR at ∼1350 nm for Type II surfaces. c Data on 6 slides from two different preparations. d Data on 32 slides from 4 different preparations. | |||
TSAc | 29 nm | 30 nm | 0.13(0.03) nmol cm−2 |
1st 6 nm(±2) | 1st 12 nm(±4) | ||
2nd 23 nm(±7) | 2nd 18 nm(±8) | ||
SSAd | 17 nm(±8) | 31 nm(±14) | 0.35(0.05) nmol cm−2 |
The stability of the grafted complexes has been checked by Cu2+ release in water from Type III surfaces. This has been done by dipping the washed and dried slides in a small volume of water, with the pH adjusted at the two representative values 4.0 and 7.0, for immersion times of 5 h and 24 h (ICP-OES analysis of copper followed). Releasing Cu2+ in water from a surface after washing it with the same solvent seems apparently contradictory, as larger volumes of water are used in the washing process and extensive Cu2+ decomplexation may be hypothesized. However metal cations actually remain on the surface after washing, as demonstrated also by total Cu2+ data in Table 2. This happens thanks to the already mentioned kinetic effect played by the surface, behaving as an “infinite dimension” substituent, that enhances the sluggishness of the release process.28 The % Cu2+ release has been calculated on the basis of total copper determined by oxidation with HNO3 on samples coming from the same preparations. To evaluate the expected release at the equilibrium, we can use the formation constants of Table 1 and calculate what would be the free Cu2+ cation if all the grafted [Cu2L]4+ complex was considered as dissolved in 3.0 mL of water (the volume used for release). Under these conditions, at pH 7.0 the 32% of copper would be free (and 68% bound to L as a mixture of [CuLH2]4+, [Cu2L]4+ and [Cu2L(OH)]3+, see ESI,† Fig. S5 for details). This fits reasonably well with the found 38(±18)% release after 24 h. The lower release at 4 h, 16(±8)%, gives instead account of the enhanced kinetic inertness of the complexation and decomplexation processes on a ligand bound to a surface by a short dangling arm. On the other hand, the same calculation carried out at pH 4.0 give 100% of expected free Cu2+. Actually we found significantly higher % of released Cu2+ with respect to pH 7.0, i.e. 60(±6)% after 4 h and 72(±17)% after 24 h. The incomplete release at lower pH was not further investigated, and can be attributed to the different environment for L between solution and surface, that may lead not only to different observed complexation constants but also to different local H+ concentration.
The photothermal behaviour of Type II and Type III slides has been measured by irradiation with a 800 nm continuous laser source (200 mW, spot diameter 1 cm) on 1 × 1 cm glass slides. These were obtained by cutting the larger slides typically used for synthesis. Thermograms (ΔT vs. t) were recorded with a thermocamera, observing in all cases an increase-plateau profile, reaching the plateau within 40 seconds, see Fig. 3. The presence of a monolayer of [Cu2L]4+ does not influence the photothermal response, as it can be seen comparing Fig. 3A (Type II surface) and Fig. 3B (Type III surface). Plateau values of 19.6(5) °C and 19.1(8) °C, respectively, were obtained. For measurements we irradiated a Type II slide, then coated it with SSA to obtain a Type III slide, then we used this for irradiation. No difference is observed despite the [Cu2L]4+ absorption at 800 nm. This is due to the dramatically different extinction coefficient of the Cu2+ d–d band (<300 M−1 cm−1, see Fig. 2C) with respect to that of the bands due to LSPR in gold nanoparticles.6,13 Finally, we irradiated glass slides of Type III (∼1 cm2) immersed in 0.500 mL of water at pH 7.0. Irradiation was carried on for 0.5 hours and the obtained water solution collected and analyzed. We observed that in this case the Cu2+ concentration in the release solution was 20% of the total Cu2+ (compared by ICP-OES Cu2+ analysis on Type III slides of the same preparation treated with HNO3 for full copper release). Such % value is higher than the % release observed after 4 h at pH 7.0 in water with no laser irradiation. Although a faster decomplexation of Cu2+ from L could also be considered, due to the increased local temperature, the 20% Cu2+ release value observed in 0.5 hours with laser irradiation, compared to the 16% release in 4 h (no laser), points towards a different explanation. In the laser irradiation case, the detachment of the whole [Cu2L]4+ complex has to be hypothesized: local T increase on GNS surface by photothermal conversion of laser irradiation has already been recently demonstrated capable of promoting breaking of gold–sulphur bonds,14 similarly to what described also on gold nanorods.30
Footnote |
† Electronic supplementary information (ESI) available: Additional SEM and TEM images, UV-Vis-NIR extinction spectra of GNS colloidal solutions and slides, distribution diagrams for Cu2+/L at micromolar concentrations. See DOI: 10.1039/c5nj03175c |
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