30 July 2021
Original Citation:
Photoacoustic ratiometric assessment of mitoxantrone release from theranostic ICG-conjugated mesoporous silica
nanoparticles
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DOI:10.1039/c9nr06524e
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COMMUNICATION
Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x
Photoacoustic ratiometric assessment of mitoxantrone release
from theranostic ICG-conjugated mesoporous silica nanoparticles
Giuseppe Ferrauto,a† Fabio Carniato,b† Enza Di Gregorio,a Mauro Botta,b and Lorenzo Tei*b
A theranostic nanosystem based on indocyanine green (ICG) covalently conjugated mesoporous silica nanoparticles (MSNs), loaded with the anticancer drug mitoxantrone (MTX) is proposed as innovative photoacoustic probe. Taking advantage from the characteristic PA signal displayed by both ICG and MTX, a PA-ratiometric approach was applied to assess drug release profile from the MSNs. After a complete in vitro characterization of the nanoprobe, a proof-of-concept study has been carried out in tumour-bearing mice to evaluate in vivo its effectiveness for cancer imaging and chemotherapeutic agent delivery.
Photoacoustic imaging (PAI) combines the properties of light and sound and it has recently emerged as a promising imaging modality with a wide range of possible applications in biomedicine.1 A typical PAI contrast agent is able to rapidly
absorb light energy and generate heat, causing thermoelastic expansion and triggering the emission of ultrasound (US) waves,2-5 detectable by US transducers. High sensitivity
exogenous imaging agents have been exploited to enhance the imaging signal.6 These consist of molecules with strong
absorption in the near-infrared (NIR) region (=700 nm–1 m), since at these frequencies the optical attenuation is weaker and a deeper tissue penetration can be achieved.
Among the NIR probes, ICG is one of the most employed for PAI applications because it is already FDA approved for human diagnosis and it has a good safety profile.7 However, its in vivo
use is hampered by two important issues, i.e. i) ICG is strongly sequestered by Human Serum Albumin (HSA),8 with a
consequent removal from the nanoprobe and ii) the photobleaching of ICG due to interaction with micro-environmental species (e.g. O2 and water of tissues).7 The
encapsulation of ICG inside nanocarriers (liposomes, micelles, lipidots, SWCN, etc…) has been exploited to enhance photostability of the molecule and prevent HSA binding,9-14
although a competition study with HSA is often omitted. Notably, we have recently shown that the encapsulation of ICG into MSNs pores increases the photoacoustic effect of ca. 400% with respect to free ICG.15 This is due to fluorescence quenching
and high photothermal conversion of the encapsulated dyes. The limitation of this approach is that HSA normally present in serum can sequester the dye, which is entrapped, but not blocked, inside the pores.
In addition, PAI shows a great potential in the imaging-guided drug delivery field, allowing noninvasive monitoring of pharmacological treatments using non-ionizing radiations. MSNs are among the nanocarriers most used in drug delivery, due to the presence of drug loadable uniform mesopores, in addition to their easy chemical functionalization and good biocompatibility.16 Recently, they have been employed to
develop theranostic nanoprobes tested for single- or multi-modality imaging approaches.17-20 At the best of our knowledge,
only few examples of theranostic PAI probes have been reported.21 Overall, they are based on the co-loading of a
PAI-active dye and the therapeutic drug in the same nanocarrier.22
Therefore, the release of the drug can be indirectly deduced by looking at the release of the PAI-active dye. However, the assumption of analogous biodistribution and release profile of the two molecules from the nanocarrier in vivo might be not always true.
The possibility to detect directly the drug by PAI is challenging. Such ideal nanosystem should contain a covalently bound PAI-active dye and a loaded chemotherapeutic agent, which is also detectable by PAI. Therefore, the aim of the present work was to design a theranostic nanoprobe in which drug delivery and release could be directly monitored by using PAI. In this way, two distinct PAI signals are expected to be generated, one reporting on the nanocarrier distribution and the other one reporting on the drug release. In light of the considerations reported above, MSNs were chosen as suitable nanocarrier, ICG dye was covalently conjugated to the MSNs and the drug mitoxantrone (MTX) was adsorbed on the surface. MTX, an anthraquinone derivative extensively investigated for the treatment of breast and prostate cancer,23 absorbs light in the
NIR range24 and its detection using optical imaging has been a Department of Molecular Biotechnology and Health Science, University of Turin,
Via Nizza 52, 10126 Torino, Italy
b Department of Science and Technological Innovation, Università del Piemonte
Orientale, Viale T. Michel 11, 15121 Alessandria, Italy
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [Experimental section, supplementary figures and schemes]. See DOI: 10.1039/x0xx00000x
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2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx
described. Nevertheless, to the best of knowledge, the direct visualization of MTX release using PAI has never been reported.
Scheme 1. Schematic illustration of the theranostic MTX-ICG-MSN
photoacoustic nanoprobe.
The structure of the nanosystem developed in this work is reported in Scheme 1. Beside ICG and MTX, MSNs surface has been also functionalized with PEG3000-COOH molecules to limit
particles aggregation when dispersed in water and to stabilize the final suspension. Briefly, MSNs were prepared by surfactant-assisted sol-gel procedure25 that allows to obtain
porous silica with particles size in the 25-50 nm range with an ordered mesopores architecture as shown by HRTEM and XRD analyses (Figure S1). MSNs were then functionalized with aminopropyl triethoxysilane followed by amide coupling of PEG3000-COOH molecules to the NH2 groups on the surface as
verified by IR spectra before and after functionalization (Scheme S1 and Figure S2). The textural properties of MSNs, before and after functionalization, were also investigated by N2
physisorption analysis (Figure S3) showing a decrease of both specific surface area and pore diameter due to the presence of the organic groups located inside the pores or on the channels entrance. The hydrodynamic diameter of PEG-NH2-MSN,
homogeneously dispersed in water at room temperature and neutral pH, is ca. 120 nm with a polydispersity index of 0.42 (Figure S4). The charge density exposed on the surface, calculated by ζ-potential, is +14.2 mV, because of the residual presence of NH2 groups (NH3+ at neutral pH) not involved in the
coupling reaction with PEG molecules.
Then, NHS-activated ICG molecules were attached to the residual amino groups exposed on the silica surface of PEG-NH2
-MSN. This strategy, compared to the simple impregnation previously adopted for the preparation of ICG-based MSNs,15
increases the chemical stability of the probe preventing release in biological fluids. The material was finally impregnated with MTX in methanol, to obtain the final theranostic probe (MTX-ICG-MSN). Other two materials based on pegylated silica, functionalized with ICG (ICG-MSN) or loaded with mitoxantrone (MTX-MSN) were also prepared as reference samples. The amount of ICG attached to the silica surface and of MTX loaded on MSNs were quantified via UV-Vis analysis as 0.3 and 38 μmol g-1, respectively.
The photophysical properties of the nanoprobes were investigated in water solution by UV-Visible and photoluminescence spectroscopy. Figure 1 shows the UV-vis
spectra of MTX-ICG-MSN in comparison to pure ICG-NHS in water at the same dye concentration (0.15 μM). An absorption at 790 nm assigned to the π → π* transitions is observed, slightly shifted to higher wavelengths with respect to the pure ICG dye, as a consequence of a different chemical environment. Furthermore, two other important bands at 609 and 661 nm are also observed and attributed to the π → π* electronic transitions of MTX drug (Figure 1A).24 The same two bands are
clearly visible in the UV-Visible spectrum of MTX (Fig. 1A) and the reference MTX-MSN sample (Figure S5).
Figure 1. (A) UV-Visible spectra in aqueous solution at neutral pH of ICG-NHS
(a), MTX-ICG-MSN (b) and MTX (c) ([ICG] = 0.15 μM; [MTX] = 11.2 μM); (B) Photoluminescence spectra of ICG-NHS (a), ICG-MSNs (b) and MTX-ICG-MSNs (c) in aqueous solution at neutral pH under excitation at 790 nm.
The excitation of the sample at 790 nm promotes an intense emission centred at 810 nm in aqueous solution. The intensity of this peak was compared to that detected for pure ICG and ICG-MSN in water at the same dye concentration (0.15 μM) (Figure 1B). Anchoring the dye to the silica surface favoured an enhancement of the quantum yield, from 2.6% for ICG-NHS to 3.4% and 5.0% for MTX-ICG-MSN and ICG-MSN samples, respectively (see ESI). Furthermore, MTX is characterized by an intrinsic photoluminescence emission when opportunely excited. Indeed, MTX-ICG-MSNs under excitation at 660 nm (at the maximum absorption of the drug) shows, along with the emission band at 810 nm, another peak at 680 nm attributed to the electronic transitions of MTX (Figure S6).
In opposite to what observed for impregnated silica with ICG,15
the chemical conjugation of the dye to the nanoparticles improves the photoluminescence efficiency of ICG and enhances the chemical stability of the probe. In fact, the analysis of the stability of the ICG-conjugated MSNs in reconstituted human serum (Seronorm™) showed no change in UV-vis spectrum over several hours (Figure S7). Conversely, the same test carried out on the ICG-impregnated MSNs sample previously reported,15 resulted in the release of the majority of
ICG molecules after 1 h, due to the high affinity of ICG for HSA. Photoacoustic properties of MSNs samples were determined on a VisualSonics Vevo Imaging Station and the behaviour of MTX-ICG-MSNs was compared to MTX-ICG-MSNs, MTX-MSNs and empty MSNs to assess the contribution of the different moieties to the photoacoustic spectrum (Figure 2). Empty PEG-NH2-MSNs are
used as control as they do not display any PA signal. ICG-MSNs display a PA peak at 810 nm in analogy to literature data for both free ICG and ICG encapsulated inside MSNs pores.7, 15 On
the other hand, MTX-MSNs show a PA peak at 700 nm characteristic of the free drug. With regard to the PA signal of ICG in MTX-ICG-MSNs particles (Figure S8), a slight
Figure 2. (A) US, (B) PAI at 700 nm, (C) PAI at 810 nm of a phantom
containing tubes filled with different solutions and (D) comparison among PAI spectra of the four MSNs formulations; 1) ICG-MSNs ([MSNs]=20mg/mL, [ICG]=6M, MTX=0) (red circles); 2) MTX-MSNs ([MSNs]=12mg/mL, [ICG]=0, MTX=452M) (black squares); 3) MTX-ICG-MSNs ([MTX-ICG-MSNs]=20mg/mL, [ICG]=6M, MTX=452 M) (blue triangles) and 4) PEG-NH2-MSNs ([MSNs]=20mg/mL, [ICG]=0, MTX=0) (green diamonds).
enhancement of PAI efficiency (ca. 75%) was observed with respect to free ICG. This enhancement is five times lower than that previously reported for ICG encapsulated inside MSNs’ pores (370%).15 On the other hand, PA efficiency of MTX is 2-3
times higher when it is free in solution compared to the drug loaded MSNs (MTX-MSNs) (Figure S9 and Figure S10). Thus, MTX-ICG-MSNs display two PA signals, at 700 nm and 810 nm provided by the presence of MTX and ICG, respectively. At the best of our knowledge, this is the first time that the PA effect of MTX has been reported. This can be useful for both developing new PAI probes and for the PAI visualization of a chemotherapeutic agent distribution in vivo. Notably, the presence of two detectable PA signals in MTX-ICG-MSNs probe allowed us to determine the ratio between the intensities at 700 and 810 nm that is expected to be constant at different probe concentrations, since it is simply due to the relative amount of ICG and MTX present in MSNs and their PA efficiency. Thus, this ratio was determined at variable concentration of MTX-ICG-MSNs (Figure S11) as 1.67 ± 0.15. It is not affected by the actual concentration of MSNs in the sample, but it is only related to the relative ICG/MTX amount in silica nanoparticles. This implies that the observation of the PA700nm/PA810nm ratio
can be useful for assessing the amount of released MTX from MSNs, since the ratio should decrease with drug release. The percentage of drug release was assessed both by using UV-vis spectrophotometry and by using PAI. UV-UV-vis experiment was carried out in phosphate-buffered saline (PBS) at 25°C. In the case of MTX-MSNs, the drug release during the first 300 min followed a linear behaviour, typical of samples containing drugs strongly interacting with the surface.26 After 15 h, the release
process reaches a plateau corresponding to a value of ca. 70% (Figure 3A). On the other hand, MTX release appears slower and reduced for MTX-ICG-MSNs. At end of the experiment, 20% of drug is released in PBS solution. The higher retention of MTX in MSNs having ICG conjugated on the surface is probably due both to the steric hindrance and non-covalent interactions with the fluorescent dye (Figure 3A).
Then, MTX release was also assessed by using PAI by means of two different approaches. In the first experiment, MTX-ICG-MSNs or MTX-MTX-ICG-MSNs were dispersed in water at the final concentration of 8 mg/mL and placed at 25°C. At variable time points (from 5 min to 48h), samples were centrifuged, and an aliquot of supernatant was collected for PAI analysis and substituted with fresh one. PA images at 700 nm and 810 nm of all collected samples were acquired. No ICG signal (PAI at 810 nm) is present for all the analysed samples (data not shown), because ICG is covalently bound to MSNs and all nanoparticles were precipitated by centrifugation. MTX signal (PAI at 700nm) is present in all collected supernatants, especially in supernatants derived from MTX-MSNs (Figure 3B-D and Figure S12). In supernatants derived from MTX-ICG-MSNs, the amount of released MTX is lower and a slower drug release kinetic is observed, in analogy to the release tests reported above. Analogous results were reported for MSNs dispersed in serum, with a slightly faster kinetic of MTX release, probably because of the presence of HSA, which could partly sequester the drug.
Figure 3: (A) Drug released from MTX-MSN (a) and MTX-ICG-MSNs (b) in PBS
solution at 25°C assessed by UV-vis spectroscopy; (B) PA signal intensity at 700 nm for MTX-MSNs (blue squares) or MTX-ICG-MSNs (blue triangle) at variable times; (C) PA spectra of MTX-MSNs’ supernatants and (D) PA spectra of MTX-ICG-MSNs’ supernatants.
In the second experiment, MTX-ICG-MSNs were suspended in water or in serum and PA images (at 700 and 810 nm) were acquired at t=0. MSNs were then left at 25°C for 24h, centrifuged and re-suspended in fresh media. Then, PA image of samples were re- acquired. Figure 4A reports US and PA images at 700 nm and 810 nm at t=0 and t=24h. Figure 4B-C report PA spectra of both samples in water and serum, respectively. In both media, the PA signal at 810 nm do not vary from t=0 to t=24h, because the total amount of ICG conjugated to MSNs is not changed and its PA effect is maintained constant. Conversely, the signal at 700 nm, provided by MTX, is strongly reduced at 24h with respect to that at t=0. In particular, quantitative insights into the release of the drug can be gained by assessing the PA700nm/PA810nm ratio. In fact, this value is
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Figure 4: (A) Representative US B-Mode image (left), PA image at 700 nm
(middle) and PA image at 810 nm (right) of MTX-ICG-MSNs (8mg/mL) in water at t=0 (sample 1) and t=24h (sample 2); (B) PA spectra of MTX-ICG-MSNs (8mg/mL) in water at variable times (t=0, 30 min, 1h, 3h, 6h and 24h); (C) PA spectra of MTX-ICG-MSNs (8mg/mL) in serum at variable times (t=0, 30 min, 1h, 3h, 6h and 24h); (D) PA signal intensity at 700 nm vs. time for MTX-ICG-MSNs in water and in serum.
carried out in water and from 1.79 to 0.69 in human serum, highlighting a substantial drug release from the MNSs.
Finally, a preclinical study was performed to investigate the in
vivo applicability of this probe for cancer theranostic studies.
PAI evaluation of a syngeneic breast tumour murine model bearing subcutaneous TS/A mammary adenocarcinoma (US B-mode image in Figure 5A, left column) was carried out in the 680-980 nm wavelengths range at three time points: before (pre-contrast), immediately after (t=0) and 30 minutes away (t=30 min) (post-contrast) from i.v. injection of MTX-ICG-MSNs (dose of 120 mg MSNs/kg in 0.2 mL of volume). In pre-contrast PA images, endogenous haemoglobin (Hb) signal was visible at 810 nm wavelength, including both the contribution of its oxygenated (oxy-Hb) and deoxygenated (deoxy-Hb) form. The presence of a significant amount of deoxygenated haemoglobin is typical of tumours at advanced development stages.27 In
post-contrast (t=0) PA image, the MTX-ICG-MSNs signal was
detectable in the tumour region (Figure 5A, second line). In particular, it can be quantified in PA spectra inside a region of interest (ROI) manually drawing the tumour contour under US B-mode image guide. Interestingly, both PA signal of MTX (=700 nm) and ICG (=810 nm) were clearly distinguishable (Figure 5A-B). An enhancement of ca. 250% was revealed by measuring PA signal intensity at =700 nm and of ca. 135% by measuring PA signal at =810 nm (Figure 6A) compared to corresponding pre-contrast images. These findings suggest that this nanoprobe was able to improve tumour detection, and that MTX was successfully delivered in tumour lesion.
Figure 5: (A) Representative US B-Mode image (left), PA image at 700 nm
(middle) and PA image at 810 nm (right) of tumour in mouse leg pre contrast (first line) and post contrast at t=0 (second line) and t=30 min (third line). (B) PA spectra of ROIs indicated with white circles in (A) (N=3).
Figure.6 (A) PA signal enhancement (%) and (B) PA700nm/PA810nm at t=0 and
t=30 min after the injection of MTX-ICG-MSNs (120 mg/kg).
In post-contrast (t=30min) PA image (Figure 5A, third line), the signal of MTX resulted significantly decreased, with a ca. 64% enhancement measured at =700 nm with respect to
pre-contrast data (Figure 6A). On the contrary, PA signal of ICG at t=
30 min post-injection shows ca. 83% enhancement with respect to pre-contrast image. It means that, from t=0 to t=30 min post-
contrast administration, there is a remarkable reduction of MTX
PA signal (contrast enhancement is reduced from 250% to 64%) and a much lower reduction of ICG PA signal (contrast enhancement is reduced from 135% to 83%) (Figure 6A). Overall, this in vivo release of MTX can be clearly highlighted by measuring the PA700nm/PA810nm ratio. This value is ca. 1.7 at t=0,
in line with the one measured in vitro for MTX-ICG-MSNs (1.67 ± 0.15) and suggesting that in vivo, at t=0 time point, MTX would be retained in MSNs. On the contrary, at t=30 min time point, the PA700nm/PA810nm ratio decreases to 0.71, in agreement with
the in vitro test of release discussed above (Figure 6B). This evidence supports the hypothesis that 30 minutes after in vivo MTX-ICG-MSNs administration, part of MTX could be released from MSNs. The kinetic of MTX release from nanocarrier and of the wash-out from the tumour region can be assessed by using this ratiometric PAI approach.
Conclusions
An innovative theranostic nanosystem has been reported, based on the use of mesoporous silica nanoparticles covalently bound to ICG dye and impregnated with mitoxantrone, as chemotherapeutic agent. The covalent binding of ICG to the MSNs surface prevents the sequestration of ICG by serum albumin, thus increasing the ICG-MSNs stability with respect to the system in which ICG was loaded into MSNs pores. The presence of ICG on MSNs surface leads to a 75% enhancement of photoacoustic efficiency with respect to the free dye and reduces the release of MTX from ca. 60% to ca. 20%.
MTX-ICG-MSNs exhibit interesting PAI properties since both ICG dye and MTX drug display evident and different PAI signals, at 810 and 700 nm, respectively. Hence, the presence of one of the two molecules or both of them can be determined by acquiring PA images. The potential application of MTX-ICG-MSNs as theranostic nanoprobe in vivo was also assessed in a preclinical murine model of cancer, testing the feasibility for detect ICG and MTX signal in the tumour region by PAI. Moreover, the ratiometric measurement of the PA effect at 700 nm and at 810 nm (PA700nm/PA810nm ratio) may allow the assessment of the
drug release from the nanocarrier, and of the wash-out kinetics of both nanosystem and drug, as suggested by the significant reduction of MTX signal 30 minutes after MTX-ICG-MSNs administration. In fact, whereas immediately after MSNs injection both PA signals are present, after the signal of is significantly reduced and that of the ICG one, which resulted only marginally decreased at the same time point.
In conclusion, the approach herein reported shows that i) theranostic MSN-based PAI nanosystems can be very useful for the simultaneous imaging and treatment of the tumour, ii) MTX can be detected and quantified in vivo by using PAI and iii) the assessment of PA700nm/PA810nm ratio can be useful to quantify
the release of MTX from the nanoparticles. Thus, a new family of nanosized PA agents, suitable for theranostic applications can be further developed for in vivo applications.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
F.C. and L.T. acknowledge Compagnia di San Paolo (CSP-2014 THERASIL Project) for funding. This project has received funding from EU-H2020-INFRADEV-2014-2015 project CORBEL, grant. n.654248 (G.F., E.D.G.). E.D.G. gratefully acknowledges Fondazione Italiana Ricerca sul Cancro (FIRC-AIRC) for her fellowship. We also acknowledge Prof. F. Cavallo, University of Torino, for providing us TS/A cell line.
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