Silica-Encapsulated Perovskite Nanocrystals for X-ray-Activated Singlet Oxygen Production and Radiotherapy Application

Multicomponent systems consisting of lead halide perovskite nanocrystals (CsPbX3-NCs, X = Br, I) grown inside mesoporous silica nanospheres (NSs) with selectively sealed pores combine intense scintillation and strong interaction with ionizing radiation of CsPbX3 NCs with the chemical robustness in aqueous environment of silica particles, offering potentially promising candidates for enhanced radiotherapy and radio-imaging strategies. We demonstrate that CsPbX3 NCs boost the generation of singlet oxygen species (1O2) in water under X-ray irradiation and that the encapsulation into sealed SiO2 NSs guarantees perfect preservation of the inner NCs after prolonged storage in harsh conditions. We find that the 1O2 production is triggered by the electromagnetic shower released by the CsPbX3 NCs with a striking correlation with the halide composition (I3 > I3–xBrx > Br3). This opens the possibility of designing multifunctional radio-sensitizers able to reduce the local delivered dose and the undesired collateral effects in the surrounding healthy tissues by improving a localized cytotoxic effect of therapeutic treatments and concomitantly enabling optical diagnostics by radio imaging.

I n the last decades, interest in nanoparticles in the biomedical field experienced a rapid growth due to the tunability of their physical and chemical properties and their rich surface chemistry that enables specific functionalization by design. 1,2 Different classes of functional nanoparticles, including metals, semiconductors, 3,4 metal/lanthanide oxides, 5,6 and organic or hybrid systems, 7,8 have found successful application in several medical branches, such as nanotherapy, diagnostics, and imaging. 9−11 Today, one of the most advanced biomedical uses of nanoparticles is offered by their strong interaction with ionizing radiation, which makes it possible to improve the effectiveness of conventional cancer treatments 12 and imaging techniques. 13 In oncological therapies, one of the most adopted medical treatments is radiotherapy (RT, ca. 50% of total cases), 14,15 a noninvasive technique typically consisting of the local release of the energy of X-rays via photoelectric effect and/or Compton scattering to stop tumor cell proliferation, either directly by damaging their DNA or indirectly by forming cytotoxic free radicals�such as singlet oxygen ( 1 O 2 ), superoxide (O 2− ) or hydrogen peroxide (H 2 O 2 )�commonly termed reactive oxygen species (ROS), upon interaction with the cellular aqueous environment. 16 Currently, in order to achieve significant therapeutic effects, patients are exposed to high doses of X-ray radiation (typically 40−60 Gy in a complete RT treatment) that carry a high risk of damaging surrounding healthy areas, due to the difficulty of finely focusing the radiotherapy exclusively on the region of interest. 17 In order to reduce X-ray exposure, several strategies have been proposed to increase the local ROS production, such as radio-stimulated photodynamic therapy and radiation catalysis. 2,18 The first is based on activating a photosensitizer responsible for the energy transfer to O 2 molecules promoting ROS production, whereas the second takes advantage of the chemical and catalytic activities of nanoparticle surfaces to enhance the generation of radiation-induced radicals by, for example, water radiolysis. 19 Metal halide nanocrystals (NCs), 20−25 both in their most common lead-based inorganic or hybrid perovskite form (APbX 3 , with A = Cs, methylammonium, formamidinium, X = Cl, Br, I) 26−28 or in lead-free alternatives, 29−31 have recently attracted substantial attention for ionizing radiation detec-tion, 32 prized for their high average atomic number (Z) that enhances the interaction probability with ionizing radiation (P i ∼ Z n , with n = 1−5 depending on the type of radiation and interaction), 33 efficient scintillation, 34−36 and strong robustness to prolonged exposure to ionizing radiation. 35 Importantly, the easy tuning of their emission spectrum from UV to NIR further makes them interesting candidates as biological markers for radio-imaging, naturally overcoming the limitations of common fluorophores to fit the near-infrared transparency window of biological tissues. 37,38 This opens up perspectives for the simultaneous application of LHP NCs in diagnostics and therapeutics, acting as X-ray biological markers to identify and target diseased areas and simultaneously as sensitizers for enhanced radiotherapy. 39 Despite such promise, very few examples of medical diagnostic and therapeutic strategies based on metal halide NCs have been proposed, 40−42 mostly because of their low stability in aqueous environment 43 64 glasses and metal oxides, 65−71 which preserve the luminescence properties of the host NCs even in harsh environments and prevent Pb dispersion in the surroundings, 29,72,73 effectively removing the constraints for the application of this class of materials in biological environments. To date, however, no study has approached the use of metal halide NCs for radiotherapy.
In this work, we aim to contribute to this endeavor by demonstrating that CsPbX 3 (X = Br, I) NCs directly synthesized inside mesoporous silica nanospheres (SiO 2 − NSs) behave as effective X-ray sensitizers for the generation of  O 2 species, boosting the effect of bare SiO 2 NSs by over 10fold. Interestingly, we found that the 1 O 2 sensitization effect is largely due to the release of secondary electrons by the CsPbX 3 −SiO 2 NSs without quenching their radioluminescence (RL) and that neither the RL nor the photoluminescence (PL) are affected by high radiation doses or by prolonged storage in an aqueous environment (even in highly acid solutions). These results, combined with the inhibition of Pb 2+ cation leakage outside the NS, made possible by the perfect sealing of the pores, open up the future possibility of implementing CsPbX 3 −SiO 2 NSs as radio-stimulated markers and therapeutic agents.
CsPbX 3 −SiO 2 NSs of different halide composition (namely, CsPbBr 3 , CsPbBr 1.5 I 1.5 , and CsPbI 3 ) were synthesized using SiO 2 NS as templates by a solid-state confined growth technique in the presence of potassium salt as sintering agent, which promotes complete collapse of the porous structure, isolating the inner CsPbX 3 NCs from the outer environment and maintaining good solubility of the NSs in water. Specifically, spherical SiO 2 NSs with diameter ∼200 nm and even distribution of internal pore dimensions were dispersed in a distilled water solution containing a proper proportion of the NC precursors (see Methods in the Supporting Information for details) and kept under stirring to favor the soaking of ions inside the pores. CsPbX 3 NCs were subsequently synthesized inside the pores by drying at 80°C to remove excess solvent followed by heating at 600°C in the presence of potassium salt (K 2 CO 3 and KI, respectively) to trigger the calcination reaction (details of the effects of the calcination temperature and conditions are reported in ref 54). Besides prompting the formation of CsPbX 3 NCs, the high temperature also favors the full collapse of the SiO 2 pores, which encloses the NCs inside the NSs and protects them from oxidation and ripening fusion. After cooling to room temperature, the CsPbX 3 −SiO 2 NSs were washed with ultrapure water several times to remove unreacted precursors and possible products formed outside the NSs, collected via centrifugation, dried at 60°C, and finally redispersed in water for further studies.
Transmission electron microscopy (TEM) images of CsPbX 3 −SiO 2 NSs are reported in Figure 1a and show spherical nanoparticles comparable to the original template NSs (see Figure S1) with a slight reduction in size due to pore collapse and subsequent shrinkage during calcination (see Figure S2), without any aggregation due to interparticle crosslinking. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and the corresponding elemental mappings (Figure 1b) of CsPbBr 3 , Figure 2. (a) Sketch of the experimental setup for the measurement of 1 O 2 production under X-ray irradiation. (b) RL spectra of CsPbBr 3 − SiO 2 , CsPbBr 1.5 I 1.5 −SiO 2 , and CsPbI 3 −SiO 2 NSs before (lines) and after (shaded areas) exposure to 20 Gy dose of X-rays. The same color code is applied to all panels. (c) SOSG PL intensity (excited at 473 nm) normalized for the initial value for pristine SiO 2 NS (black circles), CsPbBr 3 −SiO 2 (green circles), CsPbBr 1.5 I 1.5 −SiO 2 (red circles), and CsPbI 3 −SiO 2 (purple circles) as a function of X-ray exposure time. Inset: Sketch of ROS production mechanism. (d) SOSG PL intensity excited at 473 nm during the full X-ray irradiation sequence in the presence (X-ray ON) and in the absence of simultaneous X-ray irradiation (X-ray OFF) for a solution containing CsPBI 3 −SiO 2 NSs. (e) 1 O 2 production rate calculated from the linear fitting of the data in panel c. (f) X-ray mass attenuation coefficient of the investigated material systems based on the NIST database. 76 In the inset the enlargement of the mass attenuation coefficient in linear scale in the energy range of the X-rays used in our experiments is reported. CsPbBr 1.5 I 1.5 , and CsPbI 3 show that all constituent elements (Cs, Pb, Br, and I) are detected only at the NS structure, indicating that no NC remained outside the particles. Also importantly, the adopted calcination procedure maintains high particle solubility in aqueous solvent, which is a fundamental aspect to allow their applicability (as shown in Figure 1c). The crystal structure of the as-synthesized CsPbX 3 NCs inside the SiO 2 NSs and respective size distributions were studied via Xray diffraction (XRD) and TEM, as reported in Figures 1d and  S3, respectively. The XRD patterns show, in every case, a broad diffraction peak at 23°due to the contribution of amorphous SiO 2 . The diffraction peaks of the CsPbBr 3 −SiO 2 NSs at 21.36°, 26.32°, and 30.42°match cubic CsPbBr 3 structure. Consistent with their mixed halide composition, the XRD pattern of CsPbBr 1.5 I 1.5 −SiO 2 NSs shows the coexistence of cubic CsPbBr 3 , the emissive γ-phase of CsPbI 3 (peaks at 20.09°, 28.48°, and 28.92°) together with traces of the optically passive orthorhombic CsPbBr 1.5 I 1.5 δ-phase (peaks at 27.21°, 25.70°, and 31.31°); this is expected considering the thermodynamically favored crystalline transition of the CsPbI 3 γ-phase into the δ-phase below 150°C. 74 Finally, the spectrum of CsPbI 3 −SiO 2 NSs shows the γ-phase peaks and a more prominent contribution by the δ-phase. The emission properties of the CsPbX 3 −SiO 2 NSs were studied using optical and X-ray excitation, and the corresponding PL and RL spectra are reported in Figure 1e. Consistent with previous results, both the PL and RL spectra progressively shift from the green to the NIR spectral regions with increasing iodine content. 21 In all three samples, the PL spectra show the narrow peak due to excitonic emission, indicating the absence of side products or emitting defect states introduced by the confined growth in the SiO 2 NS templates. The PL quantum efficiency was found to be 55 ± 5%, 21 ± 4%, and 12 ± 3% for CsPbBr 3 , CsPbBr 1.5 I 1.5 , and CsPbI 3 , respectively.
The PL decay time of all NSs reported in Figure S4 is consistent with previous reports and features a dominant radiative fast component followed by a minor contribution due to delayed fluorescence by back-transfer from shallow traps. 35 The RL spectra are slightly red-shifted compared to the respective PL, which possibly originates from the radiative recombination of shallow emissive defect states in the proximity of energy bands typically due to halide surface vacancies as already observed in colloidal CsPbBr 3 NCs. 35,75 Next, we proceeded with validating the potential of CsPbX 3 −SiO 2 NSs as X-ray sensitizer by studying the production of the singlet oxygen ( 1 O 2 ) species in aqueous environment under X-ray irradiation. In these experiments, schematically depicted in Figure 2a, we dispersed identical concentrations of CsPbX 3 −SiO 2 NSs (2 mg/mL) with different halide composition in a phosphate buffer solution (PBS) to artificially mimic the physiological pH conditions; the same experiment was performed with bare SiO 2 NS as reference. The commercially available fluorescent probe singlet oxygen sensor green (SOSG) was used to monitor in situ the 1 O 2 evolution. In its unoxidized form, SOSG is nonemissive, whereas its endoperoxide derivative formed upon oxidation by 1 O 2 exhibits a characteristic PL band at 530 nm ( Figure S5). Therefore, the SOSG PL intensity can be used to quantify the 1 O 2 concentration during X-ray irradiation. The PL spectra of SOSG excited at 473 nm with a cw laser in PBS solutions of CsPbX 3 −SiO 2 NSs were collected during 10 min of continuous exposure to soft X-rays (E max = 20 keV) with 0.5 Gy/s dose rate. The X-ray excitation of CsPbX 3 −SiO 2 NSs simultaneously triggers the sensitization of 1 O 2 production and the NSs RL. In fact, the ability to emit RL while simultaneous sensitizing ROS production represents an important feature of our systems with respect to common radio-activated photosensitizers such as porphyrin-based assemblies that produce 1 O 2 via energy transfer to the triplet states of molecular O 2 at the expense of their luminescence. Such a feature, combined with preserving their RL emission after significant amount of radiation (as shown in Figure 2b), offers the possibility of using CsPbX 3 −SiO 2 NSs also as efficient radio-stimulated bio markers for in vitro or in vivo radio-imaging.
In Figure 2c we report the integrated intensity of the SOSG PL during the scan for the CsPbX 3 −SiO 2 NSs as well as for the control solution containing bare SiO 2 NS; on the right axis we report the respective 1 O 2 concentrations as extracted via the calibration procedure described in the Supporting Information. Notably, all solutions containing CsPbX 3 −SiO 2 NSs exhibit systematically higher 1 O 2 production with respect to bare SiO 2 NSs, with a 3-fold, 10-fold, and 13-fold enhancement along the series CsPbBr 3 −SiO 2 , CsPbBr 1.5 I 1.5 −SiO 2 , and CsPbI 3 −SiO 2 NSs, which indicates the substantial effect of the CsPbX 3 NCs on the 1 O 2 generation.
Importantly, as shown in Figure 2d for the CsPbI 3 −SiO 2 NSs (the other samples are reported in Figure S7), when the X-ray irradiation was momentarily interrupted and the solution was excited solely by the 473 nm laser, no additional 1 O 2 was created, and the trend proceeded identically only after the Xirradiation was reestablished. This is relevant since the PL of CsPbI 3 −SiO 2 NSs at 685 nm (1.81 eV) excited by the 473 nm laser source is partially resonant to the triplet state of O 2 (1.62 eV 77 ) and could, in principle, produce 1 O 2 via nonradiative energy transfer, similar to what occurs with common radioactivated photosensitizers. 78,79 The absence of 1 O 2 production without X-rays therefore indicates that the process is a direct result of the interaction of ionizing radiation with the CsPbI 3 − SiO 2 NSs with negligible mediation by its excitonic states; we note that the absence of ET despite the energy resonance could be due to the relatively large distance between the NCs and the particle surfaces imposed by the calcination procedure, as well as by the relatively fast decay time of the NC PL (4−8 ns) with respect to the micro-to-millisecond PL of sensitizer phosphorescence. 79,80 Consistent with the 1 O 2 production being dominated by the interaction probability between the CsPbX 3 −SiO 2 NSs and the X-rays, we found remarkably good correlation between the 1 O 2 production rate and the halide composition. Specifically, as shown in Figure 2e, in which we report the 1 O 2 production rate extracted from the linear fitting of the curves in Figure 2c, the NSs containing iodine-based NCs, namely CsPbBr 1.5 I 1.5 −SiO 2 and CsPbI 3 −SiO 2 , exhibited a substantially higher 1 O 2 generation rate compared to the CsPbBr 3 −SiO 2 . This trend correlates well with the mass attenuation coefficient of the systems reported in Figure 2f (calculated using the NIST database 76 and EDX analysis reported in Figure S8) in the energy range of the soft X-rays used in our experiments, which is a direct consequence of their halide composition (with the other constituents being identical). As expected based on the higher Z of I with respect to Br (53 vs 35), the mass attenuation coefficient of the NCs monotonically grows with increasing iodine content, which results in increasing release of energy in the surrounding environment and subsequently larger 1 O 2 generation rate. In fact, in the case of X-rays, the primary interactions occur by photoelectric effect or by inelastic Compton scattering, resulting in an avalanche of highly energetic secondary carriers that release their energy while traveling through a medium resulting in its ionization/excitation. Since the free path of secondary carriers is typically longer than the NS size, a substantial fraction of energy effectively escapes from the nanoparticle and is released for long distances along the ionization track, leading, in our case, to the observed strong sensitization of 1 O 2 production. 19 Notably, recent studies 81,82 demonstrated that a significant fraction of energy is deposited within the nanoparticles despite the primary interaction being shared between the nanoparticles themselves and the surrounding aqueous media. Indeed, energetic secondary charges exhibit migration ranges in most cases larger than the small nanoparticle size; consequently, a fraction of energy escapes from the nanoparticle and is released for long distances along the ionization track, directly activating the ROS production in water and the direct DNA cell damage.
Based on the promising 1 O 2 sensitization rate of CsPbI 3 − SiO 2 NSs, we further assessed their radiation resistance after 60 Gy, corresponding to the total dose that an RT patient cumulates in the entire RT treatment, as reported in Figure S9, in which the RL spectra collected before and after irradiation show that CsPbI 3 −SiO 2 retained more than 80% of its initial RL emission intensity.
Finally, to further corroborate the potential suitability of CsPbX 3 −SiO 2 NSs for X-ray stimulated applications, we assessed the risk related to the potential contamination of the environment by leakage of Pb atoms.
For this purpose, we monitored the concentration of Pb 2+ in a water solution containing CsPbX 3 −SiO 2 NSs (0.5 mg/mL) for 42 days by means of inductively coupled plasma−optical emission spectrometry (ICP-OES). The results, reported in Figure 3a, highlight that the concentration of Pb 2+ was close to the sensitivity of ICP-OES, which settles a detection limit of 10 μg/L for the Pb 2+ concentration. Considering the initial concentration of our CsPbX 3 −SiO 2 NSs, we estimate that the amount of released Pb 2+ in the monitored period is well below the 5 μg/g threshold established by the World Health Organization. To offer an illustrative comparison, if we consider a radiotherapy treatment involving the use of 10 mg of CsPbX 3 −SiO 2 for a period of time comparable to our test, the total amount of lead introduced into the body would be equal to that which would be obtained by consuming 250 g of white rice.
Finally, we monitored the optical properties of our CsPbX 3 − SiO 2 NSs in ambient atmosphere (55% humidity), water, and acid solution (1 M HCl, pH 1) in order to assess their longtime stability in conditions of potential biological interest. As shown in Figure 3b,c, nearly identical trends are observed in any condition, with nearly complete retention of the PL intensity for the CsPbBr 3 −SiO 2 and CsPbBr 1.5 I 1.5 −SiO 2 NSs and a slight (ca. 10%) loss for the CsPbI 3 −SiO 2 NSs, and the spectral properties are perfectly retained by all systems (complete spectra are reported in Figure S10).
In summary, we synthesized and studied ultrastable CsPbX 3 −SiO 2 NSs combining the strong interaction probability and scintillation features of lead halide NCs with the robustness of silica. We demonstrated that such CsPbX 3 −SiO 2 NSs dramatically sensitize the production of 1 O 2 in water under X-ray stimulation and that the generation rate correlates well with their halide composition that leads to marked differences in their mass attenuation coefficient. Our data further indicate that the 1 O 2 production is a direct result of the release of highly energetic secondary carriers in the environment and does not require quenching of their radioluminescence, thus potentially enabling their use as both therapeutic agents and radio-markers. Finally, we proved that our NSs retain their optical properties in aqueous and harsh pH conditions and under prolonged exposure to ionizing radiation. These results offer guidelines for the design of high-Z radio-sensitizers for enhancing the localized therapeutic effect of RT, reducing the delivered dose and consequent damage toward healthy tissues and thus potentially improving the quality of life of patients during and after radiological treatments.
Detailed description of the NS synthesis and experimental methods; characterization data such as MSN and NC size distribution, time-resolved PL, temperature, and stability of PL spectra in acidic and basic solutions (PDF)