# Laser-Synthesized Germanium Nanoparticles as Biodegradable Material for Near-Infrared Photoacoustic Imaging and Cancer Phototherapy

Iaroslav Belyaev, Ivan Zelepukin, Polina Kotelnikova, Gleb Tikhonowski, Anton Popov, Alina Yu. Kapitannikova, Jugal Barman, Alexey Kopylov, Daniil Bratashov, Ekaterina Prikhozhdenko, et al.

► To cite this version:

Iaroslav Belyaev, Ivan Zelepukin, Polina Kotelnikova, Gleb Tikhonowski, Anton Popov, et al. Laser-Synthesized Germanium Nanoparticles as Biodegradable Material for Near-Infrared Photoacoustic Imaging and Cancer Phototherapy. Advanced Science, 2024, 11 (20), 10.1002/advs.202307060. hal-04801074

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Submitted on 25 Nov 2024

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Laser-synthesized germanium nanoparticles as biodegradable material for near-
infrared photoacoustic imaging and cancer phototherapy

Iaroslav B. Belyaev<sup>1,2*</sup>, Ivan V. Zelepukin<sup>1,2*#</sup>, Polina A. Kotelnikova<sup>1</sup>, Gleb V. Tikhonowski<sup>2</sup>, Anton A. Popov<sup>2</sup>, Alina Yu. Kapitannikova<sup>5</sup>, Jugal Barman<sup>1</sup>, Alexey N. Kopylov<sup>2</sup>, Daniil N. Bratashov<sup>3</sup>, Ekaterina S. Prikhozhdenko<sup>3</sup>, Andrei V. Kabashin<sup>4</sup>, Sergey M. Deyev<sup>1,2,5</sup>, Andrei V. Zvyagin<sup>1,5,6#</sup>

**Abstract:** Biodegradable nanomaterials can significantly improve the safety profile of nanomedicine. Here we present germanium nanoparticles (Ge NPs) with safe biodegradation pathway as efficient photothermal converters for biomedical applications. Ge NPs, synthesised by femtosecond-laser ablation in liquids, rapidly dissolve in salt buffer with half-life of 45 min through the oxidation mechanism. The biodegradation profile of Ge nanoparticles preserved in tumour cells *in vitro* and in normal tissues in mice with a half-life as short as 3.5 days. Mild toxicity of Ge NPs was confirmed *in vivo*, showing only mild and reversible changes in the lungs and kidneys with negligible haematotoxicity. We utilised strong optical absorption of Ge in the near-infrared spectral range to demonstrate *in vivo* successful photothermal treatment of cancer cells following intravenous injection of Ge NPs. The photothermal therapy resulted in a 3.9-fold reduction of the EMT6/p adenocarcinoma tumour growth with significant prolongation of the mice survival. Excellent mass-extinction of Ge NPs (7.9 cm⁻<fnref n="1" />(g/L)⁻<fnref n="1" /> at 808 nm) enabled photoacoustic imaging of bones and tumours following intravenous and intratumoural administrations of nanomaterial. Our near-infrared-light absorbing biodegradable Ge nanomaterial appeared serendipitous for advanced theranostics.

**Introduction**

Theranostic nanomedicine has gained momentum due to its versatility, selectivity, and ability to combine imaging and therapeutic functions in one nanoagent. Photoacoustic imaging (PAI) is a hybrid technique that relies on faint ultrasound signal detection upon optical excitation [1]. PAI has shown promise for providing morphological and functional information on light-absorbing tissues and tissues labelled with contrast agents. Photothermal therapy (PTT) is a minimally invasive therapeutic regimen with proven efficiency in humans [2]. PTT relies on light-to-heat

<footnote n="1">¹ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia</footnote>

<footnote n="2">² National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow 115409, Russia</footnote>

<footnote n="3">³ Physics Institute, Saratov State University, 410012 Saratov, Russia</footnote>

<footnote n="4">⁴ Aix Marseille University, CNRS, LP3, Campus de Luminy - Case 917, 13288, Marseille Cedex, France</footnote>

<footnote n="5">⁵ Center of Biomedical Engineering, Sechenov University, 119991 Moscow, Russia</footnote>

<footnote n="6">⁶ MQ Photonics Centre, Macquarie University, 2109, Sydney, Australia</footnote>

<footnote>* These authors contributed equally.</footnote>

<footnote># Corresponding authors e-mails: zelepukin@phystech.edu (I.V. Zelepukin); andrei.zvyagin@mq.edu.au (A.V. Zvyagin)</footnote>
conversion by photosensitizers accumulated in the tumour, which causes cancer cell death locally with minimal damage to surrounding tissues. Recently, PTT using near-infrared (NIR) light excitation of gold nanoshells demonstrated promising results in clinic for prostate cancer treatment [3,4].

An ideal photosensitising agent for PAI and PTT must have large light absorption cross-section, preferably, in the optical transparency windows of biological tissues (NIR-I: 650-950 nm, NIR-II: 1100-1350 nm) [5], alongside with low toxicity, versatile surface functionalisation, and colloidal stability in biological fluids. A set of suitable photosensitising agents with high NIR-light absorbance remains exclusive to several nanomaterials, and some of them failed to prove safety for clinical applications (e.g., gold nanoparticles [6] and carbon nanotubes [7]). Slow biodegradation and long-term residence of nanoparticles in the organism tend to cause chronic and unpredictable toxicity if no effective clearance mechanism is in place [8]. NIR biophotonics champions, such as gold, iron oxide, and carbon nanoparticles can reside in mice tissues from several months to more than one year [9–11]. Two-dimensional (2D) nanomaterials exhibit exceptionally high optical mass-extinction and can be deemed suitable for PAI/PTT applications. However, except for black phosphorus and several MXenes [12,13], most types of nanosheets are not degradable due to the high crystallinity and low concentration of structural defects [14]. Thus, engineering of biocompatible and biodegradable nanoparticles for phototheranostics applications with high optical extinction in NIR range remains challenging.

Germanium belongs to the group IV material, sharing similar chemical properties with silicon whose biodegradable and optical properties have been utilised for imaging, PTT, and drug delivery applications [15]. The degradation of Ge occurs via a reaction with oxygen and hydrolysis $Ge + O_2 \rightarrow GeO_2 + H_2O \rightarrow H_2GeO_3$ [16]. Germanic acid has much higher solubility in water than silicic acid. Furthermore, unlike silicon, crystalline Ge can be dissolved in acidic environment as in lysosomes of macrophages [17]. It is not surprising that direct comparison of the dissolution rates of crystalline Si and Ge materials, shows the faster dissolution of germanium [18]. Biodegradable Germanium material is a strong optical absorber in the NIR-I and NIR-II spectral ranges, with a large Bohr radius of 24.3 nm responsible for bandgap photoluminescence in the quantum confinement regime, and effective light-to-heat conversion [19–21]. Taken together, the biodegradable, optical, and photothermal properties of Germanium are deemed very attractive for biomedical applications, provided such critical issues as efficiency and biocompatibility in vivo are addressed.

Here we present a systematic study of rapidly biodegradable Ge nanoparticles and demonstrate their promise as multifunctional theranostic agents. We synthesized Ge nanoparticles by a method of pulsed laser ablation in liquids, followed by their surface coating with albumin to improve stability and biocompatibility. Rapid hour-scale dissolution in acidic and neutral pH buffers proceeded via the oxide passivation of the nanoparticle surface. The Ge NP biodegradability pathway showed negligible toxicity in vitro and absence of chronic toxic effects after the intravenous injection in mice. Ge nanoparticles were demonstrated suitable for photoacoustic imaging and photothermal treatment of EMT6/P adenocarcinoma tumours under NIR-I laser
irradiation. We believe that the reported results speak strongly in favour of application of
Germanium nanomaterial for the next-generation photonic nanomedicine.

## Results

### Synthesis and characterisation of Ge nanoparticles

Germanium nanoparticles (Ge NPs) were synthesized by ablation of a germanium wafer in acetone with a high-energy pulsed femtosecond laser (Supplementary Figure 1). As-synthesised Ge particles were spherical in shape with partially crystalline cores, as indicated by the presence of bright Moiré fringes in high-resolution transmission electron microscopy (HR-TEM) images (Figure 1a). The particle size distribution followed a lognormal distribution, with a mean diameter of 67 ± 28 nm (Figure 1b). It is worth noting that the high-power laser ablation in the femtosecond pulsed regime yielded Ge particle size distribution [22] larger than that produced by the laser ablation operated in the nanosecond (8 – 18 nm [23]) and picosecond (3 – 43 nm [24,25]) pulsed regimes.

The Raman spectrum of Ge NPs (Figure 1c) exhibited an intense peak at 296 cm⁻¹ with a full width at half maximum of 7.2 cm⁻¹, which was attributed to the transverse mode of phonon oscillations of Ge-Ge in crystal lattice. The intensities at the characteristic shifts of GeO₂ crystalline phase (212 cm⁻¹, 261 cm⁻¹, and 440 cm⁻¹ [26]) were comparable to the background level, indicating that as-synthesized particles in acetone were not significantly oxidized. A shoulder in the range of 260 – 285 cm⁻¹ indicated the presence of a small fraction of amorphous Germanium. Amorphous phase is typical for laser-ablated nanoparticles [23,27].

### Ge nanoparticle degradation in aqueous solutions

The degradation of Ge NPs in aqueous solutions has been reported to proceed through an oxidation mechanism [21,28]. We investigated the dissolution of Ge NPs in different solutions, including distilled water (pH 5.5), phosphate-buffered saline (PBS, 0.1 M, pH 7.4), and acidic citrate buffer (0.1 M, pH 4.5). Ge NPs were incubated in solutions in 100-mg/L concentration and Ge quantities were measured at several time points by means of inductively coupled plasma mass-spectrometry (ICP-MS).

Approximately a 50 % loss of germanium mass was observed after 3 h of particle incubation in water. Then the degradation rate slowed down, resulting in a 70 % Ge release at 12 h (Figure 1d), while the remaining Ge quantity remained stable for at least 45 h. The degradation rate of Ge NPs was significantly accelerated in PBS, with 60 % and 91 % of the nanomaterial dissolved at 45 min and 5 h, respectively (Figure 1d). In citrate buffer mimicking lysosomal microenvironment, a 50 % nanomaterial loss was observed 5 h post incubation, followed by 87 % degradation after 45 h (Supplementary Figure 2). Therefore, Ge NP biodegradation was expected upon cellular endocytosis. The presence of salt ions in the solution increased the degradation rate of Germanium. This can be explained by the electrostatic interaction of Na⁺, K⁺ ions with Ge NPs, neutralizing the surface charge and promoting a release of Germanate anions. Similar mechanism has been recently proposed for silicon, a representative of the same group IV materials [29].
<figure><img src="image_2.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 1.** Synthesis and biodegradation of Ge nanoparticles. (a) HR-TEM image of Ge nanoparticles. Inset shows zoomed Moiré fringes on a particle. (b) Size distribution of Ge NPs determined from HR-TEM images. Red line shows log-normal fitting. n = 250 particles. (c) Raman spectrum of Ge nanoparticles. Red line shows Gaussian fitting. (d) Ge nanoparticle degradation in water and PBS buffer measured by ICP-MS. Data are presented as mean ± SD. Solid lines show fitting with biexponential functions. n = 3 samples. (e) Representative SEM images of Ge nanoparticles incubated in water for different periods from 0 to 48 h. (f) Evolution of optical extinction spectra of Ge NPs during their incubation in water. (g) Decay kinetics of the optical extinction, measured at 350, 500, 650, and 850-nm wavelengths. Data were normalized to the extinction intensity at 0-h time point. Solid lines show data fitting with biexponential functions.</figcaption></figure>

To investigate the mechanism of Ge NP degradation, we employed scanning electron microscopy (SEM) to observe temporal variation of the nanoparticle morphology in water. The concentration of discrete Ge NPs with a spherical shape steadily decreased over time, while particle residues formed shapeless structures after 24 hours of incubation (Figure 1e). Energy-dispersive spectroscopy coupled with SEM revealed an increase of O/Ge ratio in the Ge NPs over
time (Supplementary Figure 3). The Ge nanomaterial transformation to amorphous oxidized Germanium was completed by 48 h post incubation. These changes were confirmed by Raman spectroscopy indicated a decrease of the intensity of the peak at 300 cm⁻¹ characteristic to Ge crystalline phase, but lacking peaks of crystalline GeO₂ (Supplementary Figure 4). The high-frequency Raman shift of the crystalline Ge (from 296 to 300 cm⁻¹) corroborated previous studies of Ge NP dissolution in water [28]. These observations indicated that oxidation and hydrolysis of nanocrystalline Ge led to its amorphization and eventually dissolution. GeO₂ is known to be moderately soluble in water (10.7 g/L), while pure Ge is only soluble in strong basic or acidic solutions [30].

Optical extinction spectra of Ge NPs in solutions were acquired by UV-Vis spectroscopy to further investigate the dissolution kinetics. A monotonic decrease of the optical extinction of aqueous solutions of Ge NP was observed over time (Figure 1f, Supplementary Figure 5), which indicated the sample dissolution. Decay kinetics of the extinction was similar in the UV and visible spectral ranges (a 50 % drop at 5 h post incubation) and slowed down in the NIR-I range (Figure 1g). We infer that the larger and more oxidized Ge particles obtained after the degradation rendered the larger optical extinction cross-section in the NIR-I region. This effect can be beneficial for optical applications such as PAI and PTT, as the photothermal properties of Ge particles persist or deteriorate slowly, as the dissolution progresses.

## Colloidal stabilisation of Ge nanoparticles

To improve the biocompatibility and colloidal stability of Ge NPs in physiological conditions, we utilized a surface coating strategy by co-incubation of Ge nanoparticles with bovine serum albumin (BSA). BSA has been frequently used for nanoparticle colloidal stabilisation in drug delivery approaches due to protein hydrophilicity, high abundance in mammalian plasma, and highest structural similarity of BSA to human serum albumins among other popular xenogeneic types [31].

After the coating, a low electron density layer was observed on the surface of the Ge particles by HR-TEM attributed to the presence of organic molecules (Figure 2a). The protein quantity on the surface of NPs was measured to be 73 ± 16 mg of BSA per 1 g of particles, according to the Bradford assay. The mean hydrodynamic diameter of Ge NPs was increased from 91 ± 37 nm to 109 ± 35 nm in water likely due to dissolution of smaller nanoparticles during the coating procedure (Figure 2b). After the coating mass yield of the germanium particles was 63%, as determined by ICP-MS. Besides, stability of BSA-coated Ge NPs was markedly improved. The mean hydrodynamic diameter of BSA-Ge NPs remained unchanged in PBS for at least 90 min, while uncoated BSA-Ge NPs formed large aggregates of 600-nm size over the same period (Figure 2c). In addition, BSA-Ge NPs were characterised by a narrower ζ-potential distribution of −17 ± 6 mV (Figure 2d), indicating improved monodispersity after the coating. Energy-dispersive spectroscopy coupled with TEM revealed a large amount of nitrogen and carbon atoms likely from for proteins in BSA-Ge NPs (Figure 2e). Raman spectroscopy confirmed that the particle crystallinity was preserved after the BSA coating, while its protein content was indicated by the emergence of low-intensity bands in the amide region of 1200 – 1700 cm⁻¹, with a major peak at 1295 cm⁻¹ assigned to C-N and N-H groups vibrations (Figure 2f) [32]. Fourier transformed
infrared (FTIR) analysis of BSA-Ge NPs confirmed the surface immobilisation of BSA (Figure 2g), as indicated by the presence of characteristic to proteins amide-I (C=O stretching at 1659 cm⁻¹) and amide-II (N-H bending and C-N stretching at 1538 cm⁻¹) peaks. A Ge-O-Ge stretching band at 867 cm⁻¹ attributed to oxidized germanium was observed in the BSA-Ge NP sample and indicated dissolution propensity of the nanoparticles in water.

<figure><img src="image_9.png" /><figcaption>**Figure 2.** BSA-coating of Ge nanoparticles. (a) Representative HR-TEM image of BSA-coated Ge NPs. Scale bar – 50 nm. (b) Hydrodynamic particle size distribution of BSA-Ge NPs in water. (c) Time evolution of the mean hydrodynamic diameters of Ge NPs and BSA-Ge NPs in PBS buffer. (d) ζ-potential distribution of Ge NPs and BSA-Ge NPs in 10-mM NaCl solution. (e) Energy-dispersive X-ray mapping analysis of BSA-Ge nanoparticle. Scale bars – 10 nm. Carbon, nitrogen, and germanium elements are labelled with red, green, and blue colours, respectively. (f) Raman spectrum of BSA-Ge NPs. (g) FTIR spectra of Ge NPs and BSA-Ge NPs.</figcaption></figure>

## Photothermal properties of BSA-Ge nanoparticles

Photothermal properties of BSA-Ge NPs were evaluated in the NIR-I spectral range. BSA-coated Ge NPs displayed significantly higher optical extinction than that of uncoated Ge NPs (Figure 3a) across the near-infrared spectral range. We hypothesise that BSA-Ge NPs can have the larger size due to the dissolution of the small-sized fraction of particles during the process of BSA-coating. Theoretical modelling of light absorbance by spherical Ge NPs of size range 50 - 200 nm releaved: the larger the diameter the greater the normalised cross-section of nanoparticles in the red and NIR regions (see details in Supplementary Note 1). Note a significant enhancement of the absorbance and scattering in the NIR-I region was observed for spherical Ge particles larger than 100 nm in diameter (Supplementary Figure 6).

Photothermal heating was evaluated using an 830-nm laser operated at power of 0.7 W. BSA-Ge NPs sample in 100-µg/mL Ge concentration was heated from room temperature (24 °C) to 43.5
°C after 15 min of irradiation (Figure 3b,c). Solution with NPs concentration of 25 µg/mL was heated by 9.3 °C after 15 min (Figure 3b,c), which was in a range stipulated for medical hyperthermal treatment [33]. A comparable temperature increment has been demonstrated using crystalline 4-nm germanium nanoparticles, although 10-fold higher particle concentration was used alongside with the more favourable illumination conditions: 0.9-W laser power was employed at a wavelength of 770 nm, where the Ge absorption was greater [21]. It confirmed beneficial effects of using larger Ge NPs for phototheranostic applications.

Short-term and long-term stability of BSA-Ge NP heating was evaluated during their incubation in water in 100-µg/mL initial Ge concentration. The sample demonstrated good stability during 1-h period of cyclic heating test (Figure 3d), with no apparent photoinduced degradation. At the same time, photothermal property of the BSA-Ge NPs deteriorated during a long-term period of 1 day (Figure 3e,f). A BSA-Ge NPs was photothermally heated only by 14.3 °C above room temperature after 24-h incubation in water, which was accompanied by a decrease of the optical density at 830 nm approximately to a half of its initial value (Supplementary Figure 7). This fact confirmed degradation of BSA-Ge NPs in water, although at a slower rate compared to uncoated Ge NPs. Note that the photothermal conversion efficiency of BSA-Ge NPs changed less dramatically in the long-term stability test, i.e., from 44.4 ± 2.9 % at 0 h to 39.1 ± 3.3 % after 24 h of incubation (Figure 3g, Supplementary Figure 8). Therefore, BSA-Ge NPs remained highly efficient photothermal converters during the degradation, outperforming such recognized NIR-photothermal agents as gold and iron oxide nanoparticles in terms of conversion efficiency [34].

<figure><img src="image_10.png" /><figcaption>**Figure 3.** Photothermal properties of BSA-Ge NPs under NIR-I irradiation. (a) Extinction spectra of uncoated and BSA-coated Ge NPs normalized to Ge mass. (b) Photothermal heating/cooling kinetics and (c) corresponding peak temperature increments, following 15-min irradiation of aqueous solutions of different concentrations of BSA-Ge NPs. (d) Photothermal stability of 100-µg/mL BSA-Ge NPs during 4 20-min heating/cooling cycles. (e) Photothermal 15-min heating kinetics, (f) the corresponding peak temperature increments, and (g) the photothermal conversion efficiency of 100-µg/mL BSA-Ge NPs,</figcaption></figure>
following their incubation in water for different time periods. Heating was evaluated using 0.7-W laser irradiation at 830 nm. Data in (c,f,g) are presented as mean ± SD, n = 3 samples.

BSA-Ge NPs uptake, biocompatibility, and NIR-phototherapy in vitro

Photoconverting nanomaterials are expected to kill cancer cells by causing necrotic or apoptotic cell death upon internalization and photoactivation [35]. We thus explored cellular uptake and cytotoxicity profile of BSA-Ge NPs in different tumour cells. Particle internalization in mice adenocarcinoma EMT6/p cell line was evaluated by confocal microscopy and flow cytometry (Figure 4a,b). Accumulation of Ge particles in cells was clearly visible in bright-field mode after 45-min nanoparticle co-incubation with cells. (Supplementary Figure 10). Confocal microscopy of NPs labeled with a BSA-conjugated fluorescent tag (Cy5) revealed that particles localize in the endosomes, do not stay on the membrane, and do not penetrate into the nucleus (Figure 4a).

High refractive index of Ge NPs and significant Mie scattering allow to analyse cell uptake by side scattering (SSC) with flow cytometry, even without fluorescent labelling [36]. SSC histogram of EMT6/P cells was considerably shifted in comparison to untreated cells, while median positively correlated to a dose increase of BSA-Ge NPs (Figure 4b). Side scattering level was similar for other tested cancer lines, namely to human breast adenocarcinoma SKBR3 and human ductal carcinoma BT-474 (Figure 4b). An effective non-specific binding and uptake of BSA-coated Ge NPs to cancer cells of murine and human origin shortly after exposure gives reason to assume similar level of damage during phototherapy of human tumours.

Degradation of nanoparticles in the cells after the uptake was measured quantitatively by ICP-MS. Initial uptake of nanoparticles in 100 and 500 µg dose incubated with 10⁶ EMT6/P cells were 14 µg and 62 µg of Ge, respectively. Then we observed gradual release of Ge into the cellular environment within several days (Figure 4c). Cells exposed to the dose of 0.1 mg of Ge demonstrated a burst kinetics with release of 40% of accumulated germanium after one day and complete particle dissolution within 4 days. Increase of BSA-Ge NPs concentration to 0.5 mg slowdown dissolution with half of germanium released within first two days and 86% level of degradation at Day 4. Our data confirms possibility of rapid Ge nanoparticle degradation in acidic endosomal and lysosomal environment at the relevant doses for in vivo conditions.

Next, we investigated cytotoxicity and photothermal effect of Ge NPs under 830-nm laser irradiation at EMT6/p cell line (Figure 4d). Without irradiation, Ge NPs showed high biocompatibility with IC₅₀ of 0.86 mg/mL, calculated from the MTT test data. At the same time under NIR irradiation cytotoxicity of Ge particles greatly increased. The IC₅₀ levels for 1-min, 5-min and 15-min laser exposure were 0.16, 0.07, and 0.05 mg/mL, respectively. NIR irradiation in presence of particles led to the complete destruction of cancer cells at Ge NP dose of 0.1 mg/mL for laser exposure time of 15 min, 0.3 mg/mL for 5 min, and 1 mg/mL for only 1-min exposure (Figure 4d). It should be noted that laser treatment in absence of the nanoparticles did not significantly impact metabolic activity of cells. Analysis of proliferative activity of the cells with a clonogenic assay confirm drastic photothermal effect with BSA-Ge nanoparticles (Figure 4e). The compete tumour cells treatment with no detected formed colonies was observed for 0.1-g/L Ge concentration and 15-min laser irradiation.

While the major effect of BSA-Ge toxicity during laser irradiation is likely necrotic death due to cell overheating, an unintentional oxidative stress induction via photodynamic effect can
synergize with this pathway [37]. The changes in cellular reactive oxygen species (ROS) production over photothermal treatment were determined by a fluorogenic carboxy-H2DCFDA indicator. Analysis with flow cytometry showed strong enhancement of cell fluorescence after NIR irradiation in presence of BSA-Ge NPs (Figure 4f). We observed (5.6 ± 3.0) and (8.6 ± 0.9)-fold increase of dye fluorescence upon cells exposure to 0.1 mg/mL and 0.5 mg/mL of Ge, as compared to the untreated cells. Notably, neither separate NIR irradiation nor Ge NPs exposure didn't triggered ROS generation (Figure 4f). Oxidative stress upon photoexcitation was previously established in some other germanium-based nanomaterials [38,39]. We conclude that combined action of photothermal and photodynamic effects can ensure the effective destruction of resistant tumor cells during phototherapy with Ge NPs.

<figure><img src="image_11.png" /><figcaption>**Figure 4.** Evaluation of PTT and biodegradation of BSA-Ge NPs in vitro. (a) Representative confocal images of BSA-Ge NPs binding to EMT6/P cells. NPs were labeled with Cy5, cell nuclei were stained with Hoechst. Scale bars - 50 µm. (b) Analysis of BSA-Ge NPs binding to EMT6/P, SKBR3 and BT-474 cells by side scattering at flow cytometry. (c) Kinetics of Ge release from the EMT6/P cells after nanoparticles uptake. (d,e) MTT (d) and clonogenic assay (e) analyses of EMT6/P cells cytotoxicity dependent on BSA-Ge NPs concentration and period of 830-nm laser irradiation. (f) Analysis of reactive oxygen species generation after the NPs binding with EMT6/P cells and 830-nm laser irradiation. * - P < 0.05, ** - P < 0.01, one-way ANOVA. Data in (b-d,f) are presented as mean ± SD, n = 3 samples.</figcaption></figure>

Pharmacokinetics of BSA-Ge nanoparticles

The blood circulation and biodistribution profile of BSA-Ge NPs were measured in BALB/c mice. To this aim, nanoparticles were injected intravenously at a dose of 0.25 mg of Ge and blood samples were collected at several time points. Three animals were sacrificed after 3 h, 2 days, 7 days, 14 days, and 28 days, their main organs were collected to assay the concentration of Ge by inductively coupled plasma mass-spectrometry. The kinetics of the nanoparticle circulation in the blood demonstrated a monoexponential decay, with a half-life of t₁/₂ = 110 ± 20 s (Supplementary Figure 9). This circulation time was typical for 100-nm non-PEGylated particles administered at a low dose [40,41].
Biodistribution and biodegradation properties of BSA-Ge NPs are shown in Figure 5. After the elimination from the bloodstream, most of Ge accumulated in the liver and spleen in concentrations of 86 ± 9% ID/g and 33 ± 15% ID/g, respectively. These two organs are part of mononuclear phagocyte system and contain a number of macrophages, recognizing and removing exogenous materials from the circulation [42]. The Ge concentration in the liver gradually decreased with a half-life of t₁/₂ = 3.5 ± 0.3 days. In the spleen, the Ge concentration raised to 49 ± 18% ID/g on Day 2, followed by a gradual decrease with t₁/₂ = 3.7 ± 1.5 days. Presumably, the spleen served as a metabolic depot for Ge compounds in a short-term period, which was consistent with previous observations of Germanium fluctuations in the murine spleen within 2 days after an injection of sodium germanate [43]. In the lungs and kidneys, the highest Ge uptake was reached 3 h post injection with concentrations of 2.5 ± 1.3% ID/g and 1.8 ± 0.4% ID/g, respectively. Ge was eliminated faster in these organs compared to the liver with half-lives of 0.75 ± 0.14 days and 1.3 ± 0.3 days in the lungs and kidneys, respectively. The uptake of Ge nanoparticles by the heart and muscles was at low levels (~ 0.3% ID/g), gradually decreasing with time (Figure 5). The Ge accumulation in these organs was significantly lower compared to the other forms of inorganic Germanium, such as GeO₂ [44], Na₂GeO₃ [43], GeCl₄ [45], which were predominantly accumulated in the kidneys and lungs. A whole-body clearance of BSA-Ge nanoparticles calculated from the major organs was characterised by a half-life of 3.5 ± 0.4 days (Supplementary Figure 10), which confirmed rapid biodegradation behaviour of Ge.

<figure><img src="image_12.png" /><figcaption>**Figure 5.** Biodistribution and degradation of BSA-Ge NPs in vivo. BSA-Ge NPs were injected intravenously in BALB/c mice and Ge concentration in major organs was analysed by ICP-MS. (n = 3 animals for each point). Red lines show monoexponential fitting of the experimental data.</figcaption></figure>
NIR-photothermal therapy in vivo

BSA-coated Ge NPs were evaluated for photothermal treatment of EMT6/P adenocarcinoma in BALB/c mice. Tumour cells were inoculated subcutaneously in the flank region and monitored until the tumour volume reached 150 mm³. Then, nanoparticles at a 1-mg Ge dose were intravenously administrated in mice and tumour region was irradiated by 830-nm laser in 20 min. The animals were divided into 3 groups: animals received no treatment (“no treatment”); exposed to laser irradiation (0.4 W/cm²) for 10 min (“laser group”), treated by the same irradiation after a nanoparticle injection (“BSA-Ge NPs + laser”). The temperature of the tumour lesion was increased by 14 °C and 5 °C immediately after the PTT treatment in the tested and control groups, respectively, as measured by an infrared camera (Figure 6a,b).

The photothermal treatment with BSA-Ge NPs showed significant therapeutic benefits (Figure 6), while no severe toxicity was found associated with the decrease of the animal's body weight (Supplementary Figure 11). We observed a decrease of the mean tumour volume 2 days post-treatment from 148 ± 43 mm³ to 106 ± 61 mm³ in "BSA-Ge NPs + laser" group, while the tumour volume increased to 265 ± 67 and 277 ± 95 mm³ in the group received only laser treatment and animals without treatment, respectively. In the following period, "BSA-Ge NPs + laser" group exhibited the slower tumour growth rate than the other animals. On Day 9, the average tumour volume of these animals reached 280 ± 206 mm³, i.e., less than a 2-fold of its original volume, while the average tumour volume increased 5.5-fold to 0.8 cm³ and 7.6-fold to 1.1 cm³ in the laser-treated and non-treated groups. We emphasise that several animals treated with BSA-Ge NPs and NIR laser displayed complete tumour eradication (Figure 6d-f). Besides, a significant prolongation of the animal survival (p = 0.02) in the post-treatment period was observed after the photothermal treatment (Figure 6g). Median lifespans of the animals were 30, 34, and 54 days for the "w/o treatment", "laser", and "BSA-Ge NPs + laser" groups, respectively. The PTT treatment results demonstrated promise of BSA-Ge NPs as a photothermal agent and prompted further investigation of its applicability to photoacoustic imaging.
<figure><img src="image_13.png" /><figcaption>**Figure 6. NIR-I photothermal treatment in vivo.** (a) Kinetics of the temperature rise in the tumour during 10 min of heating using 830-nm laser exposure. n = 4 animals. (b) Time-lapse thermographic images of untreated and BSA-Ge NPs-treated mice during the PTT laser exposure. Scale bars – 2 cm. (c) The tumour volume growth kinetics after the treatment. n = 10 animals in each group. (d-f) Tumour growth kinetics of individual animals: (d) without treatment, (e) after laser treatment, (f) after BSA-Ge NPs and laser treatment, n = 10 animals in each group. (g) Kaplan-Meier plot of animal survival after the treatment, n = 10 animals in each group. In (a, c) data presented as mean ± SD. Statistical analysis was performed by ANOVA with Tukey's post-hoc test (c) or log-rank test (g). n.s. – P > 0.05, * – P < 0.05, ** – P < 0.001.</figcaption></figure>

## Photoacoustic imaging

To evaluate feasibility of utilising Ge nanoparticles for photoacoustic imaging (PAI), we examined their photoacoustic properties. PAI relies on photoinduced heating of light-absorbing agents resulting in generation of ultrasound waves via thermoelastic transduction. The photoacoustic properties of Ge NPs were measured using a TriTom tomograph under 830-nm pulsed laser excitation.

A photoacoustic signal acquired from thin capillaries filled with aqueous solutions of Ge NPs displayed linear dependence versus material concentration in a range of 20 – 500 µg/mL (Figure 7a). The lowest detection limit was estimated to be 2.3 µg/mL (confidence level 95 %). Moreover, Ge NPs in 20-µg/mL concentration provided a 4.2-fold contrast enhancement in comparison to that of distilled water.

Then we carried out PAI of BALB/c mice. An apparent photoacoustic contrast of the blood vessels and spleen was notable in the absence of BSA-Ge NPs and attributed to the strong light absorption by haemoglobin [46]. After an intravenous administration of BSA-Ge NPs, we observed an enhanced contrast in the liver and spleen, as anticipated in virtue of significant accumulation of the NPs in these organs (Figure 7b). In addition, the injected nanoparticles rendered the vertebral column and femur bones high photoacoustic contrast, as compared to almost
undetectable skeletal structures of the untreated mice (blue arrows, Figure 7b). The observed tropism of Ge NPs to the bones can be explained by the activity of bone marrow macrophages and osteoclasts in the tissue. This observation points to opportunities of photoacoustic imaging and diagnostics of bone morphology and relevant diseases.

Then we evaluated PAI of EMT6/P tumours, subcutaneously inoculated in BALB/c mice (Figure 7c,d). Bright spontaneous flashes in the tumour periphery were observed in the vicinity of the tumour vessels 3-h post intravenous administration of 1 mg of BSA-Ge NPs (blue arrows, Figure 7c). A photoacoustic contrast of the tumour was also enhanced, as seen in the axial cross-sections of the animal. Note that the PA contrast was non-uniform across the tumour, which is common situation for solid tumours, where contrast agent diffusion is hampered by the dense extracellular matrix presence. To enhance tumour contrasting, in a separate set of experiments, we injected BSA-Ge NPs of the same dosage intratumorally, followed by PAI 5-min post-administration (Figure 7d). After the injection, the tumour region displayed the highest contrast in the organism, providing better PA enhancement than that of haemoglobin abundant in the blood and other internal structures.

To push the frontiers of the emerging PAI technique, a repertoire of contrast agents suitable for in vivo imaging must be expanded. In this study we scrutinised suitability of Ge NPs as the contrast agent for PAI at 830-nm wavelength. An extinction spectrum of BSA-Ge NPs displayed a monotonic decrease in the 650 nm - 950 nm spectral range known as NIR-I biological tissue transparency window. The mass extinction measured as high as 20.1 cm⁻¹ g L⁻¹ at 650 nm dropped by ca 45 % to the value of 11.1 cm⁻¹ g L⁻¹ at 950 nm. This showed potential of nanosized germanium material as a high-quality photoacoustic imaging agent in the broad spectral range.
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Figure 7.** Photoacoustic imaging of BSA-Ge NPs. (a) Dependence of photoacoustic signal intensity on Ge NP concentration in capillaries. Red line shows linear fitting. n = 3 samples. (b) Whole body PAI of a mouse before and after intravenous injection of BSA-Ge NPs. Red arrow points to the liver, green arrow – to the spleen, blue arrows – to the bones. (c,d) PAI of EMT6/P subcutaneous tumours before and after intravenous (c) or intratumoural (d) administration of BSA-Ge NPs. Each image shows axial cross-section (left) and 3D reconstruction of the tumour region (right). Red boundaries indicate tumours. Scale bars – 5 mm. Schemes were created with BioRender.com.</figcaption></figure>

Analysis of toxicity of BSA-Ge nanoparticles at a therapeutic dose

Systemic administration of nanomaterials always raise concerns on potential toxicological
hazards and must be evaluated in terms of their cytotoxicity and tissue inflammatory response
[47]. To this aim, we investigated Ge NPs toxicological properties by measuring their interaction
with red blood cells (RBCs), analyzing relevant blood biochemistry markers, and performing
histopathological evaluation of the major organs.

Blood haemolysis and haemagglutination were evaluated using murine red blood cells. Uncoated Ge nanoparticles showed dose-dependent haemolytic activity, with RBC lysis observed at Ge NP concentrations greater than 0.2 mg/mL (Figure 8a). At the same time, BSA-coated Ge NPs did not cause RBC damage even in the 100-fold higher concentrations (20 mg/mL), and
haemolysis level was comparable to spontaneous lysis in naïve animals (Figure 8a). Haemagglutination test was carried out by employing a standard assay with U-shaped wells, where sedimentation of the aggregated RBCs formed a diffuse disk, while dispersed RBCs formed a dot on the bottom of the well. Ge and BSA-Ge NPs at concentrations of 0.02 - 20 mg/mL were incubated with RBCs for 1 h in wells. No agglutination of RBCs was observed for both uncoated and BSA-coated Ge NPs in the tested concentration range (Figure 8b). These tests indicated that BSA coating of Ge NPs prevented RBC damage. Therefore, only BSA-coated Ge NPs were used for toxicity tests in our further *in vivo* studies.

Blood biochemical analysis was performed on Days 3, 15, and 30 post intravenous administration of 1-mg BSA-Ge NPs in BALB/c mice. We analysed standard stress markers for the liver and kidney, including activity of alanine transaminase (ALT), aspartate transaminase (AST), as well as creatinine and blood urea levels. These organs were responsible for the renal and biliary excretion of BSA-Ge NPs during their degradation and were likely to be impacted. No significant alterations of ALT and AST activity of BSA-Ge NPs were revealed up to Day 30 post-injection, indicating no acute or chronic damage to the liver function (Figure 8c). Also, the blood urea level remained normal within the observation period (Figure 8c). The concentration of creatinine was elevated on Days 5 and 15, then returned to normal values on Day 30. This increase of the enzyme concentration was comparable to the levels observed in mice treated with other nanotherapeutics, including clinically approved Caelyx [48].

Histopathological evaluation showed no changes in the heart, spleen, and lung tissues, as compared to naïve mice. On Day 3 post-injection, we observed a 10% increase in the number of Kupffer cells in the liver, which is common acute reaction to the injection of nanoparticles [49]. The liver histology displayed normal tissue structure without dystrophic changes on Day 3, although signs of mild inflammation were observed. Indicators of the inflammation and cellular composition of the liver returned to normal values on Day 30. The kidney histology displayed normal organ macro- and microstructure. No signs of acute kidney tissue inflammation were found in comparison with naïve animals on Day 3, except for lymphoid infiltration of the glomeruli, mild oedema, as well as increased blood filling of the vessels. Besides, granular vacuolated cytoplasm of the glomerular epithelial cells was observed. These changes returned to normal levels on Day 30 and indicated absence of chronic toxicity caused by the administered Ge nanomaterial. We infer that the observed acute, yet reversible, stress to the kidney tissue and function was due to the massive BSA-Ge NP dissolution within the first several days after administration, followed by clearance of their degradation products via the renal filtration.
<figure><img src="image_20.png" /><figcaption>**Figure 8.** Toxicology of BSA-Ge nanoparticles after intravenous administration. (a) Haemolytic activity of uncoated and BSA-coated Ge NPs versus concentration. n = 3 samples per each data point. (b) Representative images of the haemagglutination test after RBC incubation with Ge or BSA-Ge NPs. Non-agglutinated RBCs sediment into the dot in the canter of well. (c) Activity or concentration of the blood biochemical markers: alanine transaminase (ALT), aspartate transaminase (AST), blood urea and creatinine. Grey bars show untreated animals. n = 4 samples per each data point. (d) Histological images of the major organs of untreated mice and animals after BSA-Ge NPs treatment on Days 3 and 30. Scale bars – 1 mm. Data in (a, c) are presented as mean ± SD. ** – P < 0.01, *** – P < 0.001, ANOVA with Tukey's post-hoc test.</figcaption></figure>

Discussion

We demonstrated promise of Ge NPs as a multifunctional photothermal agent with a low-toxicity degradation pathway suitable for therapeutic and diagnostic applications. Although attractive photothermal properties of Ge have been reported [21], biocompatibility of Ge material prominent in semiconductor industry remained largely unexplored, especially in the biological
tissue transparency window. We addressed this knowledge gap, paving the way for Ge NPs-based PTT and PAI applications.

Our method of pulsed femtosecond laser ablation in liquids yielded spheroidal nanoparticles with 70-nm mean size characterised by preserved partial crystallinity and endowed hydrophilicity. The nanoparticle size can be optimized during the procedure of the laser ablation *per se* or augmented by subsequent laser fragmentation [50,51]. In contrast, popular chemical methods of Ge NP synthesis by reduction of Germanium salts or thermal decomposition of organogermane precursors usually yielded sub-20-nm Ge particles by initial reaction, while the larger particles were usually aggregated or coalesced [52]. Top-down Ge NP production approaches, including high-energy ball milling yielded sub-micron Ge NPs with polydisperse size distribution characterized by irregular angled shapes [19]. Since large optical extinction of photothermal nanomaterials is pivotal for photoacoustic and photothermal applications, size tunability of Ge NP featuring strong Mie resonances in the NIR-I range (Supplementary Note 1) is important and comfortably furnished by the laser ablation method. Nanoparticles with sizes smaller than 100 nm in diameter appeared unfavorable for phototheranostic applications. Indeed, the BSA-coating of as-synthesised Ge NPs red-shifted the mean-diameter of the original particle size distribution (towards > 100 nm) and markedly enhanced the mass optical extinction (Figure 3a).

Since Germanium belongs to group IV materials, its chemical properties are expected to be similar to that of silicon. Silicon NPs have been broadly used in gene, drug delivery, and photoluminescent imaging, largely owing to their proven biodegradability [15]. This broad application scope as well as global semiconductor industry demands have led to the development of numerous NP surface modification protocols primarily based on the Stöber reaction of silanes hydrolysis [53]. We anticipate the surface modification of Ge particles will be greatly facilitated by adaptation of these protocols, and can include coating with stealth polymers, conjugation with targeting moieties and biomolecules. One needs to be cautious considering formation of solid silicon oxide layer on the particle surface can significantly slow down the degradation rate and compromise the biodegradability.

Germanium and silicon share the same degradation pathway via an oxidation and subsequent hydrolysis reactions [18]. Nevertheless, the dissolution kinetics of laser-ablated Ge NPs was faster than that of solid Si NPs [54], favoring Ge for biomedical applications. In this study, we performed first quantitative evaluation of Ge NP degradation in mammals using ICP-MS, a state-of-play in the analytical methodology, which analysed elimination of the Ge degradation products from the tissues. A whole-body clearance of Ge NPs was characterised by a half-life of $t_{1/2} = 3.5 \pm 0.4$ days. This clearance rate was considerably faster compared to that of other nanoparticles widely used in biomedicine, such as $TiO_2$ ($t_{1/2} \sim 10$ days [55]), polymer-stabilized iron oxides ($t_{1/2} > 15$ days [9]), gold ($t_{1/2} = 73$ days [56]), and soluble silica NPs ($t_{1/2} \sim 30$ days [57]). The short half-life of Ge nanoparticles in the organism alleviated risks of chronic inflammation caused by the long-term particle retention. This risk is well-known for non-biodegradable noble metal nanoparticles [58] and carbon nanomaterials [59]. Moreover, a Ge NP dissolution product, tetravalent germanium, is known to have cytotoxicity lower [60] compared to the other multivalent metal ions [61], so the
catabolism of Ge NPs is likely to exert minimal toxic stress on the organism. It has been confirmed
in our study and elsewhere [19], with no chronic toxicity observed in the tissues.

Molar extinction coefficients of Ge NPs appeared superior in comparison with the other optical
absorbers. To compare Ge nanomaterial with the other reported light-absorbing nanoparticles used
for PTT and PAI, we collated in Supplementary Note 2 their optical properties evaluated at a
popular NIR-I wavelength of 808 nm. Molar extinction of unmodified Ge was calculated as 0.57
cm⁻¹ mM⁻¹, which was comparable to the state-of-play nanomaterial, black-phosphorus quantum
dots (0.46 cm⁻¹ mM⁻¹ [62]), trailing particles composed of heavier inorganic compounds, such as
MXenes, and noble metals. However, comparing the extinction per particle, Ge NPs are at the top
of the list of nanomaterials used in phototherapy (Supplementary Table 1). The extinction per
particle of Ge NPs of 65 nm in diameter was calculated to be 4×10⁹ cm⁻¹ M⁻¹ comparable to the
best PTT performers like red-shifted plasmon Au nanorods (5.6×10⁹ cm⁻¹ M⁻¹ [63]) and Pd
nanosheets (4.1×10⁹ cm⁻¹ M⁻¹ [64]). Such high NIR-light extinction of these noble nanomaterials
enabled their successful phototheranostic applications reported to date [65]. However, since these
metal particles are not biodegradable, their application scope, including clinical applications, is
limited. Among biodegradable nanomaterials suitable for PAI and PTT, Ge NPs seem to be
superior, with the molar extinction 3-fold greater than that of the runner up, Prussian-blue
nanoparticles (1.1×10⁹ cm⁻¹ M⁻¹). It is important to note that these calculations were performed for
uncoated as-synthesised Ge NPs. BSA-coated Ge NPs featured 2-fold higher mass extinction
coefficients (Figure 3a) due to partial dissolution of the smallest fraction during the coating.
Therefore, promise of our spheroidal BSA-Ge nanoparticles for phototheranostic applications is
obvious.

Several other issues need further optimization and left behind this study. First, the size of
particles reported here seems sub-optimal. Particles larger 200 nm are rarely used for intravenous
injection due to the enhanced sequestration by macrophages. On the other hand, spherical Ge NPs
of 150-200 nm in diameter were calculated to exhibit optical extinction Mie resonances in red and
NIR spectral ranges (Supplementary Note 1). Obviously, this will enable high-contrast
photoacoustic imaging and higher efficacy PTT. Based on our theoretical modelling results, we
inferred that the contribution of particles with the diameter larger than 100 nm were dominant to
our reported treatment outcomes. Mean size and polydispersity of Ge NP colloids must be
optimized for better performance for specific in vivo scenarios.

Second, in this study, we used solid Ge nanoparticles. As such, these are poorly suited to deliver
drugs due to limited numbers of surface docking moieties. Porous Ge nano- or submicron particles
represent a promising material to realise both PAI/PTT and drug delivery carrier in one agent.
Drug release can be thermally triggered by laser radiation or a high-power radiofrequency source.
The development of porous Ge nanoparticles sized 100-200 nm represents a viable alternative to
widespread silicon-based drug delivery vehicles due to similar biocompatibility but faster
dissolution rate of Ge NPs. To this aim, one needs to consider approaches alternative to the
reported pulsed laser ablation in liquids unsuitable for production of porous nanomaterials.

**Conclusion**
In conclusion, we scrutinised an emerging photothermal converter, Germanium nanomaterial, for photoacoustic imaging and photothermal therapy applications, with an emphasis on its biocompatibility in vivo. We synthesised crystalline Ge nanoparticles by pulsed laser ablation in liquids, followed by albumin surface coating to improve compliance with biological systems. Biodegradability of Ge NPs was demonstrated by 45-min rapid dissolution in salt buffer through an oxidation mechanism, which was preserved in tumour cells in vitro, and in normal tissues in mice with a half-life of 3.5 days. Mild toxicity of Ge NPs was confirmed in cells and in vivo, showing only minor and reversible changes in the kidneys with negligible haematotoxicity. To demonstrate potential of our Ge NPs for photothermal therapy, we treated EMT6/P adenocarcinoma in mice with deeply penetrating near-infrared radiation at 808 nm and achieved a 3.9-fold reduction of the tumour growth and prolonged animal survival. A promise of Ge NPs for photoacoustic imaging was confirmed by rendering apparent photoacoustic contrast to the bones and tumours following intravenous or intratumoural NP administrations. Taken together, the demonstrated safety profile of biodegradable Ge nanomaterial makes it an optimal photothermal agent for advanced PAI/PTT theranostics, with prospects of drug delivery and photoluminescent imaging applications.

## Material and Methods

### Materials

Sigma-Aldrich (USA): bovine serum albumin (BSA); acetone; nitric acid; tween 80; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT); dimethylsulfoxide (DMSO); crystal violet; formaldehyde. Virbac (France): zoletil. Bioveta (Czech Republic): rometar. Sintez (Russia): heparin. BioXcell (USA): rat anti-mouse TER 119 antibodies. Jackson ImmunoResearch (USA): goat anti-rat IgG antibody. Gibco (UK): DMEM/F12 with GlutaMAX and HEPES, without phenol red; heat-inactivated fetal bovine serum (FBS); antibiotic-antimycotic solution; Versene solution. Invitrogen (USA): carboxy-H2DCFDA. Lumiprobe, Russia: Cyanine5 NHS-ester.

### Cells

EMT6/P cells of mouse mammary carcinoma were obtained from ECACC collection. BT474 cells of ductal carcinoma of human mammary gland, SKBR3 cells of adenocarcinoma of human mammary gland were obtained from ATCC collection. Cells were cultured in DMEM/F12 medium with GlutaMAX, supplemented with 10% FBS and antibiotic-antimycotic solution in culture flasks, under a humidified atmosphere at 5% CO₂ and 37 °C.

### Animals

Female BALB/c (10-14 weeks old, 18-22 g) mice were used in the study. Animals were obtained from Pushchino Animal Facility (Pushchino, Russia) and maintained in a vivarium at Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry. All experimental procedures were approved by the Institutional Animal Care and Use Committee (protocol № 240) of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry. The mice were housed under a 12-h light/dark cycle with free access to food and water. Animals were anaesthetised with an intraperitoneal
injection of mixture of Zoletil and Rometar in a dose 40 mg kg⁻¹ and 1.6 mg kg⁻¹, respectively, prior to any nanoparticle injections.

## Synthesis of Ge nanoparticles

The synthesis of Ge nanoparticles was carried out via femtosecond pulsed laser ablation of germanium wafer in acetone (Supplementary Figure 1). The wafer was positioned vertically along a side wall of the glass cuvette so that the distance between the target and opposite wall of cuvette was 4 mm. A radiation beam of 3 mm diameter generated by a Yb:KGW laser (1030 nm wavelength, 270 fs pulse duration, 30 µJ pulse energy, 100 kHz repetition rate; TETA 10, Avesta, Russia) was focused on the surface of the target by a F-theta lens ($f = 100$ mm). Using a galvanometer optical scanner, laser beam was set to scan a 5×10 mm area of target surface at 4000 mm/s speed for 60 min. After the ablation procedure, the obtained germanium nanoparticles were purified by centrifugation in acetone (10 min, 8000 g). To remove Ge microspheres, which can be formed during the synthesis, nanoparticles were centrifuged at 100 g for 2 min and supernatant was used for further experiments.

## Coating of Ge nanoparticles

Ge particles in acetone were washed 3 times with distilled water by centrifugation (10 min, 8000 g) to remove acetone residues. Then, particles at 1 g/L concentration were incubated in 5% BSA solution for 30 min under continuous stirring at room temperature. The non-bound proteins were removed by centrifugation (10 min, 8000 g) and particles were washed by distilled water 3 times. Mass of undissolved Ge after the coating was determined by ICP-MS. The amount of adsorbed protein after the coating procedure was measured by Bradford assay by Infinite M1000 PRO spectrophotometer at 595 nm wavelength.

## Nanoparticle characterisation

Nanoparticle size and morphology were analysed by high-resolution transmission electron microscopy (HR-TEM) with Titan Themis Z microscope (Thermo Fisher Scientific, Niderlands) coupled with Super-X energy dispersive X-ray detector. Degradation of nanoparticles was analysed by scanning electron microscopy (SEM) using Tescan MAIA 3 device (Tescan, Czech Republic) and X-act energy dispersive detector (Oxford Instruments, High Wycombe, UK) at 25 kV accelerating voltage.

Raman spectra were measured by a Confotec MR350 confocal micro-Raman spectrometer (SOL instruments, Belarus) under excitation with 633-nm laser. UV-Vis extinction spectra were recorded by Infinite M1000 PRO spectrophotometer (Tecan, Austria).

The hydrodynamic diameter and ζ-potential of nanoparticles were measured by dynamic and electrophoretic light scattering with a Zetasizer Nano ZS device (Malvern Instruments, UK). Number-size distribution was used for hydrodynamic size analysis. To measure ζ-potential nanoparticles were incubated in 10 mM NaCl for 15 min, scattering was analyzed by Smolukhowski model.

## Study of photothermal properties of BSA-Ge NPs
Photothermal heating of BSA-Ge NPs was evaluated after irradiation of solution with 830-nm laser at 0.7 W power [66]. BSA-Ge NPs were dispersed in distilled water and thoroughly sonicated. Cuvette with 2 mL of suspension was irradiated with laser for 15 min for concentration and long-term stability tests, or for 10 min for cyclic test. After these periods, solutions were naturally cooled. Temperature in liquid volume during heating and cooling was monitored by an infrared thermographic camera FLIR C3 (FLIR Systems Inc., USA). Surrounding temperature during experiments was kept at 24 ± 0.5 °C. To determine photothermal conversion efficiency, Roper's model [67] of heat transfer was used:

$$ \eta (\%) = \frac{mC(\Delta T_{max}^{NP} - \Delta T_{max}^{water})}{\tau_c I (1 - 10^{-A_{830}})}, $$

where m and C are mass and heat capacity of solution, respectively; $\Delta T_{max}^{NP}$ and $\Delta T_{max}^{water}$ are elevations of temperature for nanoparticles and water, respectively; $\tau_c$ is cooling temperature coefficient; $I$ is irradiation power; $A_{830}$ is extinction of nanoparticle solution at 1 cm path length in water. $\tau_c$ was determined from cooling kinetics $T(t)$ of solution as a slope of the negative logarithm of transfer driving force $\theta$ versus time:

$$ \tau_s = \frac{-Ln(\theta)}{t}, \\ \theta = \frac{T(t) - T_{max}^{NP}}{T_{max}^{NP} - T_s} $$

## Mass-spectrometry study of Ge NP degradation

Ge NPs were dispersed in distilled water or salt buffers at 0.1 g/L concentration. At certain time points, aliquots of 120 µL volume were taken and centrifuged for 10 min at 8000 g. Then, supernatant and residue were separated, 120 µl of 70% nitric acid were added to each sample and solutions were incubated for 15 min at 80 °C. Then samples were diluted to 10% nitric acid concentration with distilled water or salt buffer. The concentration of Ge was measured by inductively coupled plasma mass spectrometry with a NexION2000 (Perkin Elmer, USA). The mean concentration, determined over ⁷⁰Ge, ⁷²Ge, ⁷³Ge, ⁷⁴Ge and ⁷⁶Ge isotopes was used for analysis.

## Study of RBC-related toxicity

Mouse blood samples was taken by puncture of retro-orbital sinus and collected in anti-coagulating tubes. Red blood cells were washed twice with PBS via centrifugation (300 g, 5 min) and were resuspended in PBS or 1% BSA in PBS for hemolysis and hemagglutination assays, respectively. Uncoated Ge NPs were washed 4 times with water to remove acetone from particle surface; BSA-Ge NPs were used immediately after coating.

In hemolysis assay, Ge NPs were added to RBC suspension, containing 5% haematocrit, and incubated at room temperature for 30 min under mild stirring. Then, cells were centrifuged at 300 g, 5 min, and supernatant samples were collected and centrifuged at 8000 g, 5 min. The optical absorption of samples was measured at 540 nm using Infinite M1000 PRO microplate reader.
Samples of RBCs diluted in 1% Tween 80 or PBS were used as positive and negative haemolysis controls, respectively.

A hemagglutination study was performed in 96-well U-shaped plates. Ge NPs were incubated with 100 µL 1% haematocrit RBC suspension at room temperature for 1 h. RBCs in 1% BSA solution in PBS was used as negative control. For a positive control, RBCs were incubated for 10 min with rat anti-mouse RBC antibody (TER-119, 50 µg mL⁻¹), washed with PBS to remove unbound protein and were incubated with secondary goat anti-rat antibody (75 µg mL⁻¹). Agglutination of RBCs was indicated by formation of a diffuse film on the bottom of the well, while non-agglutinated RBCs sedimented in the center of the well.

## Ge NPs uptake and degradation in cells

BSA-Ge NPs were added to EMT6/P cell suspension (10⁶ cells/mL) to a final Ge concentration of 0.1 and 0.5 mg/mL. The suspensions were incubated for 60 min at 37 °C with gentle mixing every 5 min to prevent cell sedimentation. Then, cells were twice washed from unbound particles by centrifugation (5 min, 100 g) and resuspended with DMEM/F12 medium, containing 10% FBS, at final concentration of 10⁵ cells/mL. The cells were seeded at 12-well plate by 6×10⁴ at each well. Then at certain time points, cells were isolated from the medium by centrifugation (5 min, 100 g) and supernatants were collected. Samples of supernatants and sedimented cells were dissolved with 10% nitric acid in an ultrasonic bath and Ge concentration was measured by ICP-MS. The data was normalised by the measured total quantity of this element in the samples.

## In vitro cytotoxicity study

EMT6/P cells at a concentration 10⁶ cells/ml were incubated with nanoparticles for 60 min at 37 °C in phenol red free DMEM/F12 medium containing 10% FBS. 100 µL of cell suspension was irradiated with 830-nm laser at 0.91 W power for 1, 5 or 15 min. Then cells were diluted with DMEM/F12 medium containing 10% FBS, seeded into 96-well plate at a density of 5×10³ cells per well and incubated for 48 h at 37 °C under a humidified atmosphere with 5% CO₂. The cell morphology was examined under a bright-field DMI 6000B microscope (Leica Microsystems, Germany) and cell viability was measured.

To measure cytotoxicity by MTT test, cell medium was removed from the wells and 100 µL of 0.5 g/L MTT solution in serum-free medium was added to each well. After incubation for 1 h at 37 °C, MTT solution was removed and 100 µL of DMSO was added to each well. Before the measurements plates were thoroughly shaken until formazan crystals were fully dissolved. The absorbance of the samples was measured using a microplate reader at 570 nm, while 670 nm was used as a reference wavelength. The survival percentage was calculated as the signal ratio of the treated cells to the untreated ones.

To measure cytotoxicity by clonogenic assay, after the irradiation cells were seeded into 12-well plate at a density of 5 × 10³ cells per well and incubated for 7 days at 37 °C in 5% CO₂ with cell medium change every 3 days. When cells formed the colonies, cell medium was removed, and wells were subsequently washed with PBS, 50%, 70% ethanol in PBS, and cells were fixed in 96% ethanol for 15 min. Cell colonies were stained with 1% crystal violet for 30 min, rinsed under tap water to remove excess dye and allowed to dry under the air.
## Intracellular ROS measurement

EMT6/P cells at a concentration $10^6$ cells/ml were incubated with nanoparticles for 60 min at 37 °C in phenol red free DMEM/F12 medium containing 10% FBS. Then, 100 µL of cell suspension was irradiated with 830-nm laser at 0.91-W power for 5 min. Next, cells were treated with 10 µM carboxy-H2DCFDA for 30 min at 37 °C in darkness. Then, the dye fluorescence was measured using Novocyte 3000 VYB flow cytometer (ACEA Biosciences, USA) with 488-nm laser excitation and 530/30-nm band pass filter. Unstained cells and cells incubated with carboxy-H2DCFDA and 0.03% $H_2O_2$ were used as controls.

## Measurement of nanoparticle binding to cancer cells

EMT6/P, BT474 or SKBR3 cells at a concentration $10^6$ cells/ml were incubated with BSA-Ge nanoparticles for 60 min at 37 °C in phenol red free DMEM/F12 medium containing 10% FBS. Next, unbound NPs were removed by centrifugation (100 g, 5 min) and cells were resuspended in PBS. Binding of nanoparticles to cells was analysed by cell's side scattering using Novocyte 3000 VYB flow cytometer (ACEA Biosciences, USA).

For confocal microscopy visualization, BSA-Ge NPs were labeled with Cyanine5 NHS-ester according to manufacturer protocol. NPs were washed with distilled water from unbound dye by triple centrifugation (15 000 g, 15 min). EMT6/P cells were seeded at glass bottom cell slide and were allowed to grow under a humidified atmosphere with 5% $CO_2$ at 37 °C for 24 h. Next, Hoechst 33342 dye was added to cells at a final concentration of 1 µg/mL. After 1 h of incubation, cell media was removed and BSA-Ge NPs in phenol red free cell medium were added. After 4 h, unbound NPs were removed by gentle rinse with prewarmed phenol red free cell medium. Confocal microscopy was performed using LSM 980 microscope (Zeiss, Germany) under following conditions: excitation 405 nm, emission 408-501 nm for Hoechst 33342 and excitation 639 nm, emission 641-694 nm for Cyanine5 dyes.

## Pharmacokinetics study

0.25 mg of BSA-Ge NPs in 100 µL PBS were injected into retroorbital sinus of BALB/c mice. Blood samples (10-20 µL) were collected from the opposite retro-orbital sinus at 5 s, 5 min, 15 min, 30 min and 60 min after NP injection. Samples were dissolved in 0.3 mL of 20% nitric acid and diluted with 0.7 mL of water before the ICP-MS measurement. Blood bioavailability of Ge nanoparticles was quantified as average concentration of following Ge isotopes: $^{70}$Ge, $^{72}$Ge, $^{73}$Ge, $^{74}$Ge, $^{76}$Ge. Obtained concentrations were normalized to the quantity of blood in the sample, determined by $^{56}$Fe isotope.

To measure biodistribution of nanoparticles, mice were sacrificed 3 h, 2 days, 1 week, 2 weeks or 1 months after injection and major organs (liver, spleen, lungs, kidneys, heart and muscles) were collected and weighted. Then, 50-100 mg of the tissue were dissolved in a 3-fold volume of 70% nitric acid by 30-min heating at 80 °C. Then samples were diluted 10-fold with distilled water and Ge concentration was measured by ICP-MS. Data was normalized to the total measured quantity of Ge in collected tissues and presented as % ID/g.

## Toxicity analysis in vivo
BALB/c mice were injected with 1 mg dose of BSA-Ge NPs into retroorbital sinus. After 3, 15 and 30 days post-injection 200 µL of mice blood were taken from opposite retroorbital sinus and collected in K3 EDTA tubes (Microvette, Germany). Mice without treatment were used as the negative control. The activity of alanine aminotransferase, aspartate aminotransferase, as well as concentrations of creatinine and blood urea nitrogen were measured by Infinite M1000 PRO spectrophotometer (Tecan, Austria) using commercial kits (Olvex, Russia).

For histopathological analysis, mice were sacrificed at 3 and 30 days post-injection via cervical dislocation. Major organs (liver, spleen, lungs, kidneys, heart) were collected, incubated for 24 h in 4% PBS-based formaldehyde solution, dehydrated in a rising ethanol series and embedded in paraffin. Then, tissues were cutting into sections of 4-µm thickness and were stained with haematoxylin and eosin. Slices were photographed using Leica DMI6000B microscope equipped with Leica DMC2900 camera (Leica Microsystems, Germany).

## Photothermal therapy in vivo

10⁶ EMT6/P cells in 100 µL PBS were inoculated subcutaneously into flank region of BALB/c mice. In 6 days, mice with single tumours with an average volume of 150 mm³ were chosen for the experiments. The mice were anesthetized, placed on an orbital shaker and fixed by a tape lent. Mice were divided into 3 groups: without treatment; with irradiation by 830-nm laser for 10 min; and with treatment by 1 mg of BSA-Ge NPs and the same laser irradiation. Treatment was performed 20 min after BSA-Ge NPs injection. The laser beam was focused on the tumour volume and power density was 0.4 W/cm². Mice were slowly rotated during the irradiation to prevent skin burning. Measurements of tumor volume temperature and whole-body thermographic images were performed with FLIR C3 camera. The tumour size was measured every day after the treatment by a mechanical caliper and the volume was estimated by the ellipsoid formula. The survival analysis was performed according to Kaplan-Meier model by monitoring animals every 1-2 days.

## Photoacoustic tomography

The TriTom tomograph (PhotoSound Technologies, Inc., Houston, TX, USA) was used to obtain photoacoustic images of Ge NP solutions and mice. For in vitro experiments, the aqueous solutions of BSA-Ge nanoparticles were placed in the thin capillaries and the capillary ends were sealed with silicon. For in vivo experiments, mice were anesthetized and shaved with a trimmer and depilatory cream. Then, the mouse was placed in a frame holder and paws were clamped with soft fasteners. The head of the mouse was hooked with its front teeth on a horizontal holder, air was supplied directly through the tube. All measurements were carried out using deionized water as the acoustic coupling media. Both Ge NP solutions and mice were submerged in deionized water filled tank alongside the ultrasonic transducer array. The pulsed OPO laser (PhotoSonus, Ekspla, Lithuania) tuned to 830-nm wavelength was used for photoacoustic excitation. The sample was rotated with 10°/s velocity during the measurement. The 3D photoacoustic images of both Ge NP solutions and mice were reconstructed with 0.1×0.1×0.1 mm voxel size using TriTom software (PhotoSound Technologies, Inc., Houston, TX, USA).

## Statistical analysis
All experiments were performed at least in triplicates. Data are presented as mean ± standard
deviation (SD). Significant differences were determined using one-way ANOVA with Tukey post-
hoc test. Significant differences of the survival curves were evaluated using log-rank test.

**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Contributions**

I.B.B. and I.V.Z. designed experiments. G.V.T., A.A.P. and A.V.K. synthesized particles. I.B.B., I.V.Z., J.B. and A.N.K. coated and characterised nanoparticles and their degradation. P.A.K. performed cell experiments. A.Yu.K., I.B.B. and J.B. characterize in vivo toxicity. I.B.B., I.V.Z. performed in vivo experiments and PTT treatment. I.B.B., D.N.B. and E.S.P. performed photoacoustic study. A.V.K., S.M.D. and A.V.Z. supervised the study. I.B.B., I.V.Z., A.V.Z., A.V.K. and S.M.D. analysed data, wrote, and corrected manuscript.

**Acknowledgements**

The work was supported by the Ministry of Science and Higher Education of the Russian Federation, agreement no. 075-15-2020-773.

**Data availability statement**

The main data supporting the results of this study are available within the paper and its Supplementary Information.

**References**

[1] Srivalleesha Mallidi, Geoffrey P. Luke, Stanislav Emelianov, Photoacoustic imaging in cancer detection, diagnosis, and treatment guidance, Trends in Biotechnology 29 (2011) 213–221.
https://doi.org/10.1016/j.tibtech.2011.01.006.

[2] H.S. Han, K.Y. Choi, Advances in Nanomaterial-Mediated Photothermal Cancer Therapies: Toward Clinical Applications, Biomedicines 9 (2021) 305. https://doi.org/10.3390/biomedicines9030305.

[3] A.R. Rastinehad, H. Anastos, E. Wajswol, J.S. Winoker, J.P. Sfakianos, S.K. Doppelapudi, M.R. Carrick, C.J. Knauer, B. Taouli, S.C. Lewis, A.K. Tewari, J.A. Schwartz, S.E. Canfield, A.K. George, J.L. West, N.J. Halas, Gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study, Proc. Natl. Acad. Sci. U. S. A. 116 (2019) 18590–18596.
https://doi.org/10.1073/pnas.1906929116.

[4] J.M. Stern, S.V.V. Kibanov, E. Sazykina, J.A. Schwartz, S.C. Gad, G.P. Goodrich, Initial Evaluation of the Safety of Nanoshell-Directed Photothermal Therapy in the Treatment of Prostate Disease, International journal of toxicology 35 (2016). https://doi.org/10.1177/1091581815600170.

[5] D.C. Sordillo, L.A. Sordillo, P.P. Sordillo, L. Shi, R.R. Alfano, Short wavelength infrared optical windows for evaluation of benign and malignant tissues, J. Biomed. Opt 22 (2017) 45002.
https://doi.org/10.1117/1.jbo.22.4.045002.

[6] A. Sani, C. Cao, D. Cui, Toxicity of gold nanoparticles (AuNPs): A review, Biochemistry and Biophysics Reports 26 (2021) 100991. https://doi.org/10.1016/j.bbrep.2021.100991.
[7] M. Ema, M. Gamo, K. Honda, A review of toxicity studies of single-walled carbon nanotubes in laboratory animals, Regulatory Toxicology and Pharmacology 74 (2016) 42–63.
https://doi.org/10.1016/j.yrtph.2015.11.015.

[8] G. Yang, S.Z.F. Phua, A.K. Bindra, Y. Zhao, Degradability and Clearance of Inorganic Nanoparticles for Biomedical Applications, Adv. Mater. 31 (2019) 1805730.
https://doi.org/10.1002/adma.201805730.

[9] I.V. Zelepukin, A.V. Yaremenko, I.N. Ivanov, M.V. Yuryev, V.R. Cherkasov, S.M. Deyev, P.I. Nikitin, M.P. Nikitin, Long-Term Fate of Magnetic Particles in Mice: A Comprehensive Study, ACS Nano 15 (2021) 11341–11357.
https://doi.org/10.1021/acsnano.1c00687.

[10] J. Kolosnjaj-Tabi, Y. Javed, L. Lartigue, J. Volatron, D. Elgrabli, I. Marangon, G. Pugliese, B. Caron, A. Figuerola, N. Luciani, T. Pellegrino, D. Alloyeau, F. Gazeau, The One Year Fate of Iron Oxide Coated Gold Nanoparticles in Mice, ACS Nano 9 (2015) 7925–7939.
https://doi.org/10.1021/acsnano.5b00042.

[11] M. Zhang, Y. Tahara, M. Yang, X. Zhou, S. Iijima, M. Yudasaka, Quantification of whole body and excreted carbon nanohorns intravenously injected into mice, Adv. Healthc. Mater. 3 (2014) 239–244.
https://doi.org/10.1002/adhm.201300192.

[12] J. Shao, H. Xie, H. Huang, Z. Li, Z. Sun, Y. Xu, Q. Xiao, X.-F. Yu, Y. Zhao, H. Zhang, H. Wang, P.K. Chu, Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy, Nat Commun 7 (2016) 1–13.
https://doi.org/10.1038/ncomms12967.

[13] B. Zhou, H. Yin, C. Dong, L. Sun, W. Feng, Y. Pu, X. Han, X. Li, D. Du, H. Xu, Y. Chen, Biodegradable and Excretable 2D W1.33 C i-MXene with Vacancy Ordering for Theory-Oriented Cancer Nanotheranostics in Near-Infrared Biowindow, Adv. Sci. (Weinh) 8 (2021) e2101043.
https://doi.org/10.1002/advs.202101043.

[14] F. Xia, J. Lao, R. Yu, X. Sang, J. Luo, Y. Li, J. Wu, Ambient oxidation of Ti3C2 MXene initialized by atomic defects, Nanoscale 11 (2019) 23330–23337.
https://doi.org/10.1039/c9nr07236e.

[15] J.-H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti, S.N. Bhatia, M.J. Sailor, Biodegradable luminescent porous silicon nanoparticles for in vivo applications, Nature Mater 8 (2009) 331–336.
https://doi.org/10.1038/nmat2398.

[16] M.F. Ehman, K. Vedam, W.B. White, J.W. Faust, Characterisation of thin surface films on germanium in various solvents by ellipsometry, J Mater Sci 6 (1971) 969–973.
https://doi.org/10.1007/BF00549947.

[17] W. Pugh, CXCIX.—Germanium. Part IV. The solubility of germanium dioxide in acids and alkalis, J. Chem. Soc. 0 (1929) 1537–1541.
https://doi.org/10.1039/jr9290001537.

[18] S.-K. Kang, G. Park, K. Kim, S.-W. Hwang, H. Cheng, J. Shin, S. Chung, M. Kim, L. Yin, J.C. Lee, K.-M. Lee, J.A. Rogers, Dissolution Chemistry and Biocompatibility of Silicon- and Germanium-Based Semiconductors for Transient Electronics, ACS Appl. Mater. Interfaces 7 (2015) 9297–9305.
https://doi.org/10.1021/acsmi.5b02526.

[19] Pengbo He, Guo Chen, Mengling Huang, Lili Jing, Wen Wu, Hao-Chung Kuo, Chang-Ching Tu, Sung-Liang Chen, Biodegradable germanium nanoparticles as contrast agents for near-infrared-II photoacoustic imaging, Nanoscale 15 (2023) 11544–11559.
https://doi.org/10.1039/D3NR01594G.

[20] N. Shirahata, D. Hirakawa, Y. Masuda, Y. Sakka, Size-Dependent Color Tuning of Efficiently Luminescent Germanium Nanoparticles, Langmuir 29 (2013) 7401–7410.
https://doi.org/10.1021/la303482s.
[21] Timothy N. Lambert, Nicholas L. Andrews, Henry Gerung, Timothy J. Boyle, Janet M. Oliver, Bridget S. Wilson, Sang M. Han, Water-Soluble Germanium(0) Nanocrystals: Cell Recognition and Near-Infrared Photothermal Conversion Properties, Small 3 (2007) 691–699.
https://doi.org/10.1002/smll.200600529.

[22] M. Priyadarshini, J.N. Acharyya, I.S. Deo, G.V. Prakash, Strong third-and higher-order nonlinear optical studies on femtosecond laser ablated Germanium metalloid nanoparticle colloid in the wide spectral domain (450–1500 nm), Optics & Laser Technology 156 (2022) 108515.
https://doi.org/10.1016/j.optlastec.2022.108515.

[23] S. Vadavalli, S. Valligatla, B. Neelamraju, M.H. Dar, A. Chiasera, M. Ferrari, N.R. Desai, Optical properties of germanium nanoparticles synthesized by pulsed laser ablation in acetone, Front. Phys. 2 (2014) 57. https://doi.org/10.3389/fphy.2014.00057.

[24] Marina Rodio, Alice Scarpellini, Alberto Diaspro, Romuald Intartaglia, Tailoring of size, emission and surface chemistry of germanium nanoparticles via liquid-phase picosecond laser ablation, J. Mater. Chem. C 5 (2017) 12264–12271. https://doi.org/10.1039/C7TC01992K.

[25] Y. Hamanaka, M. Iwata, J. Katsuno, Fabrication of oxidation-resistant Ge colloidal nanoparticles by pulsed laser ablation in aqueous HCl, Appl. Phys. A 123 (2017) 1–7.
https://doi.org/10.1007/s00339-017-1044-9.

[26] T. Sato, J. Suda, Lattice Dynamics and Temperature Dependence of the Linewidth of the First-Order Raman Spectra for Sintered Hexagonal GeO₂ Crystalline, Journal of the Physical Society of Japan 67 (1998) 3809. https://doi.org/10.1143/JPSJ.67.3809.

[27] A.A. Fronya, S.V. Antonenko, N.V. Karpov, N.S. Pokryshkin, A.S. Eremina, V.G. Yakunin, A.Y. Kharin, A.V. Syuy, V.S. Volkov, Y. Dombrovska, A.A. Garmash, N.I. Kargin, S.M. Klimentov, V.Y. Timoshenko, A.V. Kabashin, Germanium Nanoparticles Prepared by Laser Ablation in Low Pressure Helium and Nitrogen Atmosphere for Biophotonic Applications, Materials 15 (2022) 5308.
https://doi.org/10.3390/ma15155308.

[28] Y. Hamanaka, M. Iwata, J. Katsuno, Fabrication of oxidation-resistant Ge colloidal nanoparticles by pulsed laser ablation in aqueous HCl, Appl. Phys. A 123 (2017) 1–7.
https://doi.org/10.1007/s00339-017-1044-9.

[29] F.K. Crundwell, On the Mechanism of the Dissolution of Quartz and Silica in Aqueous Solutions, ACS Omega 2 (2017) 1116–1127. https://doi.org/10.1021/acsomega.7b00019.

[30] M. KRISHNA MURTHY, H. HILL, Studies in Germanium Oxide Systems: III, Solubility of Germania in Water, Journal of the American Ceramic Society 48 (1965) 109–110.
https://doi.org/10.1111/j.1151-2916.1965.tb11811.x.

[31] K.A. Majorek, P.J. Porebski, A. Dayal, M.D. Zimmerman, K. Jablonska, A.J. Stewart, M. Chruszcz, W. Minor, Structural and immunologic characterization of bovine, horse, and rabbit serum albumins, Mol. Immunol. 52 (2012) 174–182. https://doi.org/10.1016/j.molimm.2012.05.011.

[32] V.J. Lin, J.L. Koenig, Raman studies of bovine serum albumin, Biopolymers 15 (1976) 203–218.
https://doi.org/10.1002/bip.1976.360150114.

[33] Y. Cao, Q. Ren, R. Hao, Z. Sun, Innovative strategies to boost photothermal therapy at mild temperature mediated by functional nanomaterials, Materials & Design 214 (2022) 110391.
https://doi.org/10.1016/j.matdes.2022.110391.

[34] Ana Espinosa, Jelena Kolosnjaj-Tabi, Ali Abou-Hassan, Anouchka Plan Sangnier, Alberto Curcio, Amanda K. A. Silva, Riccardo Di Corato, Sophie Neveu, Teresa Pellegrino, Luis M. Liz-Marzán, Claire
Wilhelm, Magnetic (Hyper)Thermia or Photothermia? Progressive Comparison of Iron Oxide and Gold Nanoparticles Heating in Water, in Cells, and In Vivo, Advanced Functional Materials 28 (2018) 1803660. https://doi.org/10.1002/adfm.201803660.

[35] J.R. Melamed, R.S. Edelstein, E.S. Day, Elucidating the Fundamental Mechanisms of Cell Death Triggered by Photothermal Therapy, ACS Nano 9 (2015) 6–11.
https://doi.org/10.1021/acsnano.5b00021.

[36] R. M. Zucker, K. M. Daniel, E. J. Massaro, S. J. Karafas, L. L. Degn, W. K. Boyes, Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence, Cytometry Part A 83 (2013) 962–972. https://doi.org/10.1002/cyto.a.22342.

[37] Valentina Guerrero-Florez, Stelia C. Mendez-Sanchez, Olga A. Patrón-Soberano, Vicente Rodríguez-González, Diana Blach, Fernando Martínez O., Gold nanoparticle-mediated generation of reactive oxygen species during plasmonic photothermal therapy: a comparative study for different particle sizes, shapes, and surface conjugations, J. Mater. Chem. B 8 (2020) 2862–2875.
https://doi.org/10.1039/D0TB00240B.

[38] Min Ge, Haiyan Guo, Ming Zong, Zhixin Chen, Zhuang Liu, Han Lin, Jianlin Shi, Bandgap-Engineered Germanene Nanosheets as an Efficient Photodynamic Agent for Cancer Therapy, Angewandte Chemie International Edition 62 (2023) e202215795. https://doi.org/10.1002/anie.202215795.

[39] Q. Li, H. Fan, Y. Xu, M. Liu, J. Liu, L. Xu, M. Zou, Q. Cheng, Y. Zhang, T. Liang, L. Shi, X. Hu, L. Wang, Z. Wang, NIR-responsive hollow germanium nanospheres mediate photothermal/photodynamic therapy and restrain immunosuppression to cooperatively eradicate primary and metastatic tumors, Chemical Engineering Journal 458 (2023) 141314.
https://doi.org/10.1016/j.cej.2023.141314.

[40] I.V. Zelepukin, A.V. Yaremenko, M.V. Yuryev, A.B. Mirkasymov, I.L. Sokolov, S.M. Deyev, P.I. Nikitin, M.P. Nikitin, Fast processes of nanoparticle blood clearance: Comprehensive study, Journal of Controlled Release 326 (2020) 181–191. https://doi.org/10.1016/j.jconrel.2020.07.014.

[41] B. Ouyang, W. Poon, Y.-N. Zhang, Z.P. Lin, B.R. Kingston, A.J. Tavares, Y. Zhang, J. Chen, M.S. Valic, A.M. Syed, P. MacMillan, J. Couture-Sénécal, G. Zheng, W.C.W. Chan, The dose threshold for nanoparticle tumour delivery, Nat. Mater. 19 (2020) 1362–1371. https://doi.org/10.1038/s41563-020-0755-z.

[42] Y.-N. Zhang, W. Poon, A.J. Tavares, I.D. McGilvray, W.C.W. Chan, Nanoparticle–liver interactions: Cellular uptake and hepatobiliary elimination, Journal of Controlled Release 240 (2016) 332–348.
https://doi.org/10.1016/j.jconrel.2016.01.020.

[43] G. Rosenfeld, Studies of the metabolism of germanium, Archives of Biochemistry and Biophysics 48 (1954) 84–94. https://doi.org/10.1016/0003-9861(54)90308-2.

[44] C.-H. Lin, T.-J. Chen, Y.-L. Hsieh, S.-J. Jiang, S.-S. Chen, Kinetics of germanium dioxide in rats, Toxicology 132 (1999) 147–153. https://doi.org/10.1016/s0300-483x(98)00147-4.

[45] I. Velikyan, G. Antoni, J. Sörensen, S. Estrada, Organ biodistribution of Germanium-68 in rat in the presence and absence of 68GaGa-DOTA-TOC for the extrapolation to the human organ and whole-body radiation dosimetry, Am. J. Nucl. Med. Mol. Imaging 3 (2013) 154–165.

[46] Jun Xia, Muhammad R. Chatni, Konstantin I. Maslov, Zijian Guo, Kun Wang, Mark A. Anastasio, Lihong V. Wang, Whole-body ring-shaped confocal photoacoustic computed tomography of small animals in vivo, JBO 17 (2012) 50506. https://doi.org/10.1117/1.JBO.17.5.050506.
[47] Y. Yang, Z. Qin, W. Zeng, T. Yang, Y. Cao, C. Mei, Y. Kuang, Toxicity assessment of nanoparticles in various systems and organs, Nanotechnology Reviews 6 (2017) 279–289.
https://doi.org/10.1515/ntrev-2016-0047.

[48] M.P. Nikitin, I.V. Zelepukin, V.O. Shipunova, I.L. Sokolov, S.M. Deyev, P.I. Nikitin, Enhancement of the blood-circulation time and performance of nanomedicines via the forced clearance of erythrocytes, Nat Biomed Eng 4 (2020) 717–731. https://doi.org/10.1038/s41551-020-0581-2.

[49] M. Bartneck, T. Ritz, H.A. Keul, M. Wambach, J. Bornemann, U. Gbureck, J. Ehling, T. Lammers, F. Heymann, N. Gassler, T. Lüdde, C. Trautwein, J. Groll, F. Tacke, Peptide-Functionalized Gold Nanorods Increase Liver Injury in Hepatitis, ACS Nano 6 (2012) 8767–8777.
https://doi.org/10.1021/nn302502u.

[50] A V Kabashin, M Meunier, Femtosecond laser ablation in aqueous solutions: a novel method to synthesize non-toxic metal colloids with controllable size, J. Phys.: Conf. Ser. 59 (2007) 354.
https://doi.org/10.1088/1742-6596/59/1/074.

[51] A. Al-Kattan, Y.V. Ryabchikov, T. Baati, V. Chirvony, J.F. Sánchez-Royo, M. Sentis, D. Braguer, V.Y. Timoshenko, M.-A. Estève, A.V. Kabashin, Ultrapure laser-synthesized Si nanoparticles with variable oxidation states for biomedical applications, J. Mater. Chem. B 4 (2016) 7852–7858.
https://doi.org/10.1039/c6tb02623k.

[52] Dimitri D. Vaughn II, Raymond E. Schaak, Synthesis, properties and applications of colloidal germanium and germanium-based nanomaterials, Chem. Soc. Rev. 42 (2013) 2861–2879.
https://doi.org/10.1039/C2CS35364D.

[53] P. van Der Voort, E.F. Vansant, Silylation of the Silica Surface A Review, Journal of Liquid Chromatography & Related Technologies 19 (1996) 2723–2752.
https://doi.org/10.1080/10826079608015107.

[54] I.V. Zelepukin, E.A. Mashkovich, N.A. Lipey, A.A. Popov, V.O. Shipunova, O. Yu. Griaznova, M.S. Deryabin, V.V. Kurin, P.I. Nikitin, A.V. Kabashin, M.I. Bakunov, S.M. Deyev, A.V. Zvyagin, Direct photoacoustic measurement of silicon nanoparticle degradation promoted by a polymer coating, Chemical Engineering Journal 430 (2022) 132860. https://doi.org/10.1016/j.cej.2021.132860.

[55] D. Elgrabli, R. Beaudouin, N. Jbilou, M. Floriani, A. Pery, F. Rogerieux, G. Lacroix, Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection, PLoS One 10 (2015) e0124490.
https://doi.org/10.1371/journal.pone.0124490.

[56] J.H. Lee, J.H. Sung, H.R. Ryu, K.S. Song, N.W. Song, H.M. Park, B.S. Shin, K. Ahn, M. Gulumian, E.M. Faustman, I.J. Yu, Tissue distribution of gold and silver after subacute intravenous injection of co-administered gold and silver nanoparticles of similar sizes, Arch Toxicol 92 (2018) 1393–1405.
https://doi.org/10.1007/s00204-018-2173-4.

[57] E. Choi, S. Kim, Surface pH buffering to promote degradation of mesoporous silica nanoparticles under a physiological condition, Journal of Colloid and Interface Science 533 (2019) 463–470.
https://doi.org/10.1016/j.jcis.2018.08.088.

[58] R. Mohammadpour, M.A. Dobrovolskaia, D.L. Cheney, K.F. Greish, H. Ghandehari, Subchronic and chronic toxicity evaluation of inorganic nanoparticles for delivery applications, Advanced Drug Delivery Reviews 144 (2019) 112–132. https://doi.org/10.1016/j address of the paper.
https://doi.org/10.1016/j.ambr.2019.07.006.

[59] C.S. Lim, D.W. Porter, M.S. Orandle, B.J. Green, M.A. Barnes, T.L. Croston, M.G. Wolfarth, L.A. Battelli, M.E. Andrew, D.H. Beezhold, P.D. Siegel, Q. Ma, Resolution of Pulmonary Inflammation
Induced by Carbon Nanotubes and Fullerenes in Mice: Role of Macrophage Polarization, Front. Immunol. 11 (2020) 1186. https://doi.org/10.3389/fimmu.2020.01186.

[60] E. Sabbioni, S. Fortaner, S. Bosisio, M. Farina, R. Del Torchio, J. Edel, M. Fischbach, Metabolic fate of ultratrace levels of GeCl(4) in the rat and in vitro studies on its basal cytotoxicity and carcinogenic potential in Balb/3T3 and HaCaT cell linesdagger, J. Appl. Toxicol. 30 (2010) 34–41. https://doi.org/10.1002/jat.1469.

[61] A. Manke, L. Wang, Y. Rojanasakul, Mechanisms of nanoparticle-induced oxidative stress and toxicity, Biomed Res. Int. 2013 (2013) 942916. https://doi.org/10.1155/2013/942916.

[62] T. Fan, Y. Zhou, M. Qiu, H. Zhang, 2018. Black phosphorus: A novel nanop platform with potential in the field of bio-photonic nanomedicine. Journal of Innovative Optical Health Sciences 11, 1830003. https://doi.org/10.1142/S1793545818300033.

[63] Guanglei Fu, Wei Liu, Shanshan Feng, Xiuli Yue, Prussian blue nanoparticles operate as a new generation of photothermal ablation agents for cancer therapy, Chem. Commun. 48 (2012) 11567–11569. https://doi.org/10.1039/C2CC36456E.

[64] X. Huang, S. Tang, X. Mu, Y. Dai, G. Chen, Z. Zhou, F. Ruan, Z. Yang, N. Zheng, Freestanding palladium nanosheets with plasmonic and catalytic properties, Nature Nanotech 6 (2011) 28–32. https://doi.org/10.1038/nnano.2010.235.

[65] Zhuoqian Lv, Sijia He, Youfu Wang, Xinyuan Zhu, Noble Metal Nanomaterials for NIR-Triggered Photothermal Therapy in Cancer, Adv. Healthc. Mater. 10 (2021) 2001806. https://doi.org/10.1002/adhm.202001806.

[66] A. Paściak, A. Pilch-Wróbel, Ł. Marciniak, P.J. Schuck, A. Bednarkiewicz, Standardization of Methodology of Light-to-Heat Conversion Efficiency Determination for Colloidal Nanoheaters, ACS Appl. Mater. Interfaces 13 (2021) 44556–44567. https://doi.org/10.1021/acsami.1c12409.

[67] D.K. Roper, W. Ahn, M. Hoepfner, Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles, The Journal of Physical Chemistry C 111 (2007) 3636–3641. https://doi.org/10.1021/jp064341w.