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

Abstract Biodegradable nanomaterials can significantly improve the safety profile of nanomedicine. Germanium nanoparticles (Ge NPs) with a safe biodegradation pathway are developed as efficient photothermal converters for biomedical applications. Ge NPs synthesized by femtosecond‐laser ablation in liquids rapidly dissolve in physiological‐like environment through the oxidation mechanism. The biodegradation of Ge nanoparticles is preserved in tumor cells in vitro and in normal tissues in mice with a half‐life as short as 3.5 days. Biocompatibility of Ge NPs is confirmed in vivo by hematological, biochemical, and histological analyses. Strong optical absorption of Ge in the near‐infrared spectral range enables photothermal treatment of engrafted tumors in vivo, following intravenous injection of Ge NPs. The photothermal therapy results in a 3.9‐fold reduction of the EMT6/P adenocarcinoma tumor growth with significant prolongation of the mice survival. Excellent mass‐extinction of Ge NPs (7.9 L g−1 cm−1 at 808 nm) enables photoacoustic imaging of bones and tumors, following intravenous and intratumoral administrations of the nanomaterial. As such, strongly absorbing near‐infrared‐light biodegradable Ge nanomaterial holds promise for advanced theranostics.


Supplementary Note S1. Modeling of light interaction with Ge nanoparticles of variable sizes
The interaction of electromagnetic waves with a homogenous Ge spherical NPs in a water medium was modeled using the Mie solution to Maxwell's equations in Matlab software (2018b version).The extinction, scattering, and absorption cross-sections () for a given wavenumber k of light were defined by the following expressions (equations 1-3): (1) (2) whith Mie coefficients (equations 4,5): The Ricatti-Bessel functions (equations 6,7): The dimensionless parameters for calculation (equations 8-10): The Bessel functions of the first ( ν ) and second (  ) order were calculated in the Matlab environment using the built-in functions.The refarctive indices for Ge and water were taken from refractiveindex.info. [1]The calculated cross section for each particle size was normalized to the volume of space occupied by the single nanoparticle with the largest diameter in a simulation (200 nm).

Supplementary Note S2. Comparison of NIR-light extinction and biodegradability of phototheranostic nanomaterials
Notable inorganic nanomaterials, reported for photothermal or photoacoustic biomedical applications, were compared for the values of optical extinction at commonly used NIR-I wavelength of 808 nm based on the published data.We searched for numerical values of extinction coefficient per mass As usually only one of these quantities could be derived, the following formula was applied for conversion: where   is Avogadro's constant,  is bulk density, and   is expected volume of an average particle.
To calculate   , morphology and average characteristic dimensions of particles were considered from corresponding references and the following shape approximations were applied: sphere for nanoparticles (NPs), square plate for nanosheets/nanoplates (2D) and QDs, cylinder for nanorods (NRs).For materials with no explicitly reported dimensions, calculation of molar extinction was not applied (NA).The data on the extinction and biodegradability of the selected materials are presented in the Table S1.Materials belonging to categories A, B, C, and D are highlighted in blue.

Figure
Figure S3.a) SEM images (top) and corresponding energy-dispersive element mapping (down) of Ge NPs incubated in water for different time periods.Scale bars -1 µm.Elements of Ge and O are marked with yellow and blue colors, respectively.b) Evolution of energy-dispersive spectra of Ge NPs incubated in water for different time periods.The intensities were normalized to the peak signal of Ge L-line.

Figure S4 .
Figure S4.Evolution of Raman spectra of Ge NPs incubated in water for different time periods.

Figure S5 .
Figure S5.Photographs of Ge NP solutions incubated in water for different periods.Initial concentration of nanoparticles was 100 mg L -1 .

Figure S6 .
Figure S6.Colloidal stability of BSA-Ge NPs in PBS and DMEM/F12 over time.a,b) Evolution of

Figure S7 .
Figure S7.ICP-MS evaluation of BSA-Ge NPs degradation in PBS buffer and DMEM/F12 medium.

Figure
FigureS8shows modeled data of normilized cross-sections for light extinction, absorption, and

Figure
Figure S8.a) Normalized extinction, b) absorption and c) scattering efficiencies of Ge NPs of different sizes.

Figure
Figure S9.a) Photograph of photothermal heating experimental set-up and b) evolution of thermal map during irradiation in 100 μg mL -1 nanoparticle solution.Dashed line contours the cuvette's profile.

Figure S10 .
Figure S10.a,b) Change of optical extinction of BSA-Ge NP water solution with concentration (a) and time (b) at wavelength of 830 nm.For time-dependence, 100 µg mL −1 is a starting concentration of particles.c) Photographs of same nanoparticle solution during irradiation with 830 nm laser immediately after dispersion in water and after 5 and 24 h.

Figure S11 .
Figure S11.a) Cooling kinetics of BSA-Ge NPs in long-term photothemal stability test.Starting temperatures correspond to the moment of switching off the laser irradiation.b) Corresponding dependencies of logarithm of driving force θ on cooling time.Blue lines show linear fitting with slope .

Figure S12 .
Figure S12.Bright-field microscopy of EMT6/P cell culture before and after treatment with BSA-Ge NPs at concentration 1 g L −1 .Scale bar -100 µm.

Figure S13 .
Figure S13.Representative flow cytometry histograms, showing side scattering shift of EMT6/P cells after their incubation with different concentrations of BSA-Ge nanoparticles.

Figure S14 .
Figure S14.a) Evolution of Rhodamine B absorbance spectra under dark incubation; heating at 60 °C; NIR laser exposure at 1-W power; dark incubation with 1 g L -1 Ge NPs; incubation with 1 g L -1 Ge NPs under NIR laser exposure at 1-W power.b) Plot of the Rhodamine B dye degradation kinetics.

Figure S15 .
Figure S15.Blood circulation kinetics of BSA-Ge NPs in mice determined by ICP-MS.n = 3 animals for each time-point.The red line shows monoexponential fitting.

Figure S16 .
Figure S16.Cumulative elimination kinetics of BSA-Ge NPs from all measured organs (liver, spleen, lungs, kidneys, and heart) of mice.n = 3 animals for each time-point.The red line shows monoexponential fitting.

Figure S17 .
Figure S17.Dynamics of mouse body weight change after photothermal therapy.The number of animals in each group started from n = 5 on day 0 and maximally decreased to n = 3 with time due to the animals' death.The data are presented as group mean ± standard deviation.
Also, the extinction coefficient per mole of molecular substance ( 808  ) was calculated by relation with molar mass м [g mol -1 ]:  808  =  808  * м.The analyzed nanomaterials were also classified based on their ability to biodegrade in physiologically-relevant environments, claimed in the reference reporting optical extinction or elsewhere.The following classification from high (A) to low (E) biodegradation potential was used: A -Degradable in water; B -Degradable in aqueous solutions at physiological pH; C -Enzymatically biodegradable; D -Degradable enzymatically or at physiological pH after the surface coating;E -Commonly considered non-biodegradable or no proof of biodegradability was found.

Table S1 .
Comparison of extinction coefficients at 808 nm and biodegradation properties of different nanomaterials.