Large photoacoustic effect enhancement for ICG confined inside MCM-41 mesoporous silica nanoparticles

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Cite this:Nanoscale, 2017, 9, 99 Received 21st October 2016, Accepted 19th November 2016 DOI: 10.1039/c6nr08282c www.rsc.org/nanoscale

Large photoacoustic e

ffect enhancement for ICG

con

fined inside MCM-41 mesoporous silica

nanoparticles

†

Giuseppe Ferrauto,

Fabio Carniato,

Enza Di Gregorio,

Lorenzo Tei,*

Mauro Botta

and Silvio Aime*

Indocyanine green was encapsulated inside the pores of pegylated amino-functionalized MCM-41 Mesoporous Silica Nanoparticles (ICG-MSNs). In addition to a greater stability and a decrease of tox-icity, the photoacoustic e_{ffect of ICG-MSNs increases by nearly} 400% compared to free ICG due tofluorescence quenching and high photothermal conversion of the encapsulated dyes. Upon i.v. administration in tumor-bearing mice, an overall photoacoustic enhancement ofca. 25% was measured in the tumor region.

ICG has been extensively used in medical diagnosis for a number of applications and, above all, in preclinical optical studies.1 _{In particular it has been employed for rapid blood} volume measurements,2 _{for angiography}3 _and ophthal-mology.4_{In addition, its application has also been proposed} for guided surgery5 _{as well as in sentinel lymph node} detec-tion.6_{More recently, it has been demonstrated that ICG is also} an efficient dye for photoacoustic imaging (PAI).7

PAI has emerged in the last few years as an innovative imaging technique that can provide biomedical, structural and functional information on specific regions of interest.8_{It e} ffec-tively merges the ultrasound’s advantages (spatial resolution, penetration depth, etc.) with the optical properties of absorb-ing molecules naturally occurrabsorb-ing in tissues or externally admi-nistered, such as sensitivity and possibility to visualize con-temporarily different molecules.8_{Numerous endogenous} con-trast molecules (hemoglobin, melanin, lipid, water)9_{have been}

successfully exploited as PAI contrast agents to gain physiologi-cal information about healthy and pathologiphysiologi-cal tissues (vascu-lar volume, oxygen content, etc.). More recently, the develop-ment of exogenous contrast agents has emerged as a promi-nent field of research.10,11_{Typically, an efficient PAI contrast} agent should be characterized by: (i) biological compatibility and lack of toxicity; (ii) water solubility and a sufficiently long half-life compatible with an efficient excretion from the body; (iii) high light-to-ultrasound energy conversion; (iv) suitability to chemical modifications in order to endow the agent with targeting properties; and (v) ease of preparation and good stability.10–12 Many PAI agents have been already proposed. They belong to four main classes, namely: (i) molecular dyes; (ii) fluorescent proteins; (iii) plasmonic surface resonance noble metal nanoparticles and (iv) non-plasmonic nano- or microparticles.10_{Molecular dyes typically employed in optical} imaging have been translated to optoacoustic applications (e.g. Methylene Blue, Congo Red, NIRF molecules, ICG, etc.) showing, in general, a good sensitivity without significant negative side effects.10_{In particular, ICG is already approved} by the FDA for human application and is undoubtedly safer than plasmonic surface resonance metals (Au, Ag, etc.); however, the in vivo application is hampered by a rapid protein binding, fast clearance, and instability under physiologically relevant conditions.13 _{Thus, the encapsulation of ICG inside} nanocarriers appears to be a suitable way to improve its per-formance as a molecular imaging probe. Several exogenous nanocarriers have been tested including ICG-encapsulated micelles,14_{ICG-doped PLGA,}15_{HSA (Human Serum} Albumin)-ICG,16 _{ICG-loaded liposomes,}17 _{lipidic particles (lipidots)}18 and ICG-coated single-walled carbon nanotubes.19 _Typically, they show enhanced photostability and better bioavailability but not an increased photoacoustic effect with respect to the free dye. Analogously, the encapsulation of ICG20 _{as well as} other NIR cyanine dyes21_{inside silica NPs has been} demon-strated to protect the molecules from degradation.

Since MSNs are highly stable and versatile, we designed a new PAI probe by loading ICG dyes inside the pores of pegy-lated MCM-41 MSNs (Fig. 1) with the aim to increase both the †Electronic supplementary information (ESI) available: Methods (synthesis of

mesoporous support (MSNs), impregnation of ICG in the silica nanoparticles and PEG anchoring, tools for MSN characterization, ICG-loaded liposomes, cell cultures, mouse models, in vitro and in vivo photoacoustic experiments), sup-plementary results (HR-TEM micrographs, DLS hydrodynamic radius, surface ζ-potential, X-ray pattern, pore diameter distribution, thermogravimetric ana-lysis, UV-visible, FT-IR, photoluminescence spectra of MSNs, and relative PAI signal of ICG-MSNs with a different ICG loading) and supplementary references. See DOI: 10.1039/c6nr08282c

a_{Department of Molecular Biotechnology and Health Sciences, University of Turin,}

Via Nizza 52, 10126 Turin, Italy. E-mail: [email protected]

b_{Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte}

Orientale“A. Avogadro”, Viale T. Michel 11, 15121 Alessandria, Italy. E-mail: [email protected]

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stability of the dye and its photoacoustic effect.22_{In this work,} MCM-41 NPs were prepared through a typical sol–gel pro-cedure by using a block-copolymer and a surfactant-assisted co-assembly method.23

Physico-chemical characterization of MSNs (HR-TEM, XRD, N2physisorption analysis) is reported in the ESI (Fig. S1–S3†). The results show a system composed of spherical particles with size in the 20–50 nm range and a regular hexagonal pore array of 2.5 nm diameter. Then, the surface of the MSNs was decorated with amino propyl groups24in order to: (i) allow the grafting of PEG-COOH molecules on the surface and (ii) promote the ionic interaction between the ammonium ions on the MSNs and the sulfonates of the ICG molecules, as pre-viously reported in the case of ICG-loaded trimethyl-ammonium modified MSNs.25 The amount of –NH2 groups present on the surface was calculated by thermogravimetric analysis under oxygen flow and resulted to be 1.3 mmol g−1 (Fig. S4†).

In the second step, organo-modified MCM-41 NPs were sus-pended in a MeOH solution of ICG (0.07μM) for 8 h at room temperature to allow the impregnation of the dye into the silica channels. After washing with MeOH, the ICG loading in ICG-MSNs was 0.80 μmol g−1 (ca. 25 ICG molecules per par-ticle, see ESI† for details), assessed by measuring the amount of free dye in the washing medium using UV-visible spec-troscopy at 785 nm (Fig. S5†). To evaluate the result of the amount of dye loaded in MSN pores on the photoacoustic effect, samples containing different amounts of ICG (1.7 and 3.7μmol g−1) were also prepared.

Finally, a fraction of the–NH2groups exposed on the silica surface was reacted with polyethylene glycol (PEG5000-COOH) via amide-coupling methods to improve particle stability and to confer a stealth character towards the immune system.26 PEG moieties located on the MSNs’ external surface ensured good compatibility in aqueous solution preventing particle aggregation. The NPs appeared substantially monodispersed in water with a hydrodynamic diameter of ca. 60 nm (Fig. S6†) and a surfaceζ-potential of +21.4 mV at 25 °C (Fig. S7,† data unchanged with respect to the MSN-NH2 nanoparticles). The IR spectrum of the pegylated NPs showed an additional absorption at 1650 cm−1, assigned to the stretching of amide groups formed by the coupling of PEG to the surface

(Fig. S8†). It is worth noting that, no release of ICG molecules was detected during the reaction.

ICG-MSNs were also characterized by UV-visible and photo-luminescence spectroscopy. As reported in Fig. 2A, ICG-MSNs show a main absorption at 810 nm, promoted by the π → π* transitions, slightly shifted to higher wavelengths with respect to the isolated dye, as a consequence of a different local chemi-cal environment. The excitation of the ICG-MSN at 780 nm promotes a strong emission in the 810–840 nm range (Fig. S9†). A quenching of the fluorescence emission for ICG-MSNs was observed when compared to that of equivalent concentrations of the free dye resulting in a ca. 2.2 fold decrease of quantum yield (Table 1).

The fluorescence quenching of ICG loaded inside the MSN pores was comparable to that of gold nanostructures27and can be attributed to dye self-aggregation.28

Remarkably, photodegradation (bleaching tests) of ICG inside silica channels is almost completely absent after 1 hour of light irradiation (Fig. 2B and Table 1) and no release in solu-tion of ICG was detected during the experiment. This behavior appears very similar to that measured for pegylated liposomes incorporating ICG, prepared as previously reported.17In con-trast, the free dye showed a degradation of ca. 60% under iden-tical experimental conditions.

It is known that, when ICG molecules are loaded inside nanocarriers, their accessibility to water is limited and their stability is enhanced.17,20 The cyanine photobleaching is mainly caused by the generation of reactive oxygen species (ROS) triggered by photoexcitation of fluorophores.29 Therefore, molecular oxygen dissolved in water (0.3 mM) has a central role and when ICG is free in aqueous solution its

stabi-Fig. 1 A schematic view of the distribution of ICG molecules inside functionalized mesoporous silica NPs.

Fig. 2 (A) UV/Vis spectra of ICG (black curve), ICG-MSNs (red curve) and ICG-liposomes (blue curve) in aqueous solution (ICG concentration for all samples is 1.70 μM). (B) Photostability tests on ICG (black), ICG-MSNs (red) and ICG-liposomes (blue) in aqueous solution (ICG con-centration = 1.70μM), irradiated at λ = 750 nm.

Table 1 Comparison of stability, optical and photoacoustic properties between ICG, ICG-MSNs and ICG-liposomes

System ICG stability (irradiation at λ = 750 nm, 1 h) Quantumefficiency Photoacoustic effect ICG 35% 2.58%31 ₁ ICG-MSNs 90% 1.13% +370% ICG-liposomes 100% 5.50% +25%

lity is strongly affected. Conversely, when such molecules are partially shielded from water solvent their stability is strongly enhanced. In the herein described MSNs, the ICG molecules are confined inside the silica pores where the water diffusion is markedly slowed down,30thus reducing the negative effect of ROS on ICG stability.

It is noteworthy that, the absorption spectrum and the photodegradation pattern of ICG-MSNs and ICG-liposomes are almost identical (Fig. S9† and Fig. 2B). Nevertheless, in the case of ICG loaded into liposomes, the quantum efficiency in water resulted higher than that of free ICG (Table 1), as also observed in the case of ICG encapsulated into micellar systems.31

Moreover, ICG-MSNs are stable in physiological media for over 24 h (Fig. S10†), whereas release tests carried out in Seronorm™ indicated a progressive loss of ICG from the silica NPs, likely caused by the strong affinity of ICG for HSA.32_The test was performed by dispersing 6 mg of ICG-MSN in 3 mL of a Seronorm™ solution and monitoring the UV-Vis absorbance of the dye over time. 10 wt% and 60 wt% of ICG molecules were released after 15 min and 1 h, respectively. This result highlights the need of a further improvement of the system to prevent release of ICG from MSN’s pores before an extended in vivo translation.

Prior to ICG-MSNs in vivo application, cell toxicity studies were carried out in vitro in J774A.1 murine macrophage cul-tures. 1.5 × 106cells were plated in 6 cm Petri dishes overnight and successively incubated for 3 h at 37 °C, 5% CO2 in the presence of different amounts of ICG-MSNs (in a range corres-ponding to 0–20 μM of ICG). As a control, J774A.1 cells were incubated in the presence of free ICG at the same concen-trations present in MSNs. After the incubation, cells were extensively washed with fresh PBS, detached by means of a scraper and counted by using a trypan blue exclusion assay. Cell viability was evaluated with respect to control unlabeled samples. As reported in Fig. 3A, cell viability is ca. 100% up to a [ICG] = 6μM for both free ICG and ICG-MSNs. At higher con-centrations, ICG-MSNs showed a lower toxicity with respect to free ICG (e.g. 11% vs. 27% at 20μM, Fig. 3A).

The experiments were repeated in quadruplicate and mean ± SD values are reported. A two-tailed Student’s t-test was per-formed. Furthermore, to double check count results, the cells were centrifuged and the pellets suspended into 250μL of PBS

and lysed by sonication (30 s, power 30%, 20 kHz). Then, the total protein content was evaluated by the Bradford assay and used as an indirect evaluation of cell number in each sample. The same samples were used to evaluate the uptake efficiency. In cell lysates, the fluorescence was measured by using a Horiba FluoroMax Fluorimeter and normalized by considering the relative cell number of each sample. Normalized fluo-rescence intensity (a.u.) was plotted against the concentration of ICG-MSNs or free ICG values in the incubation medium (Fig. 3B), showing that ICG entrapped inside MSNs is less efficiently sequestered by macrophages with respect to free ICG (at the tested concentrations). Consequently, ICG-MSN blood availability is longer and they can be detected in vivo for a longer time compared to free ICG.

Then, photoacoustic properties of ICG-MSNs were analyzed by using a VisualSonics Vevo 2100 LAZR Imaging Station (VisualSonics, Inc., Toronto, Canada) equipped with a LZ250 transducer operating at 21 MHz. For in vitro PAI, ICG-MSN sus-pensions were loaded onto plastic capillaries surrounded by 1% agarose gel (Fig. 4A–C). The acquisition of PAI spectra shows that these nanoparticles display two PA peaks at 725 and 810 nm (Fig. 4D). For this system the detection threshold (i.e. the minimum concentration of probe needed to have a PA signal higher than the background, herein represented by MSNs w/o ICG) corresponds to ca. 5–6 μM of ICG (Fig. 4E). Remarkably, when ICG is localized inside MSNs the PAI efficiency increases by almost 4 times compared to that of free ICG (+370% signal intensity; ca. one order of magnitude con-sidering the area under curve, Fig. 5).

Fig. 3 (A) Cell viability of murine macrophages upon labeling with free ICG (blue) or ICG-MSNs (red); (B) normalized_{fluorescence intensity of} cell lysates upon labeling with free ICG (blue) or ICG-MSNs (red).

Fig. 4 (A) US image, (B) PAI (_{λ = 810 nm) and (C) merge of US and PA} images of a phantom composed of plastic tubes_{filled with ICG-MSNs at} variable concentrations corresponding to [ICG] = (1) 0_{μM, (2) 2.3 μM, (3)} 5.8_{μM, (4) 11.7 μM and (5) 17.5 μM. (D) PA spectra of ICG-MSNs at} vari-able concentrations; (E) PA signal_{vs. concentration. The dotted line} rep-resents the detection threshold.

The strong intermolecular interaction among ICG mole-cules inside MSN pores, responsible for the fluorescence quenching, translates into photothermal conversion by non-radiative decay and thus to an increase of the photoacoustic effect. This explanation finds support in the behavior of ICG-loaded liposomes that generate a higher quantum yield (Table 1) and then do not increase the PAI effect with respect to free ICG.

Interestingly, a clear role in the generation of an efficient PAI effect is played by the amount of ICG molecules present in the MSNs. It is known that the molar absorption extinction coefficient (ε) of ICG in solution decreases at high concen-trations as a consequence of aggregation phenomena,13in our case, an analogous effect is observed by comparing three ICG loadings in MSN (0.8, 1.7 and 3.7 μmol g−1, respectively, Table S1†). When the amount of ICG encapsulated inside MSN pores is too high, the ε is very low and the PAI response is weak as a consequence of the low absorption capability (Fig. S11†). Finally, as a proof of concept of the in vivo applica-bility of this probe, mice bearing subcutaneous tumors (US image in Fig. 6A) were i.v. injected with ICG-MSNs (at the dose of 120 mg MSNs per kg) and PA images of the tumor region were acquired ca. 20 min post injection. For this system, as previously reported,25a hepatic accumulation and excretion is expected.

Upon ICG-MSNs administration, an overall PAI (λ = 810 nm) signal enhancement was observed inside the tumor region (Fig. 6C and D). A quantitative calculation of the signal enhancement was carried out in a small portion of the tumor (ROI manually drawn and highlighted by dotted line in Fig. 6C and D) that corresponds to ca. 25%. The differences between pre and post ICG-MSN administration were clearly detectable by looking at the PA spectra inside dotted ROI. Before the administration of the nanoparticles, a typical PA spectrum of hemoglobin was observed (Fig. 6B). In particular, a large amount of deoxygenated hemoglobin was present, as typical of

tumors at late development stages. Conversely, the PA spec-trum acquired after the administration of the photoacoustic nanoprobe, showed the expected signal due to the presence of ICG-particles entrapped in the MSN channels.

Conclusions

In summary, it has been shown that ICG can dramatically change its optical and PA properties depending on its locali-zation (e.g. in solution or inside MSN pores or within the inner cavity of a liposome) and on its actual concentration inside the pores of the nanoparticle. Thus, the confinement of ICG mole-cules into MSNs allowed to obtain a PAI effect much greater (ca. 400% higher) than free ICG and ICG-loaded liposomes. This property is likely to be associated with the strong inter-action of ICG molecules inside MSNs that quenches the fluo-rescence by increasing the photothermal conversion of the absorbed light. However, we observed that, when the amount of encapsulated ICG is too high (e.g. in the 3.7 μmol g−1 sample), the molar absorption extinction coefficient becomes too low thus making the system not suitable to generate a PAI signal.

Moreover, the reported ICG-MSNs efficiently stabilize ICG and reduce cell toxicity and uptake by macrophages, being thus more suitable for in vivo experiments. Further studies will be directed to improve the ICG confinement into the silica pores, thereby preventing partial release when the particles are in contact with serum proteins (HSA). Then, with the opti-mized system, a combination of the enhanced photostability and photoacoustic signal with the ability of MSNs to be

func-Fig. 6 (A) US image of the tumor region; (B) photoacoustic spectra of a tumor ROI pre (black) and post (red) administration of ICG-MSNs; PA images of the tumor region (λ = 810 nm) pre (C) and post (D) adminis-tration of ICG-MSNs.

Fig. 5 (A) US image of plastic tubes containing PBS, ICG 14 μM or ICG-MSNs 12 mg mL−1(corresponding to 14μM of ICG); (B) PA image (λ = 810 nm) of the same phantom; (C) merge of US and PA images; (D) PA spectra of samples.

tionalized with targeting vectors or other imaging reporters, would allow the future development of efficient molecular imaging PAI probes.

Acknowledgements

We gratefully acknowledge the support of AIRC (IG-GRANT, S. A.), MIUR (PRIN 2012; project 2012SK7ASN_002) and Compagnia di San Paolo (CSP-2014 THERASIL Project, F. C., L. T.). G. F. and E. D. G. were supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro AIRC) fellowships.

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