Theranostic Inorganic-Organic Hybrid Nanoparticles with a Cocktail of Chemotherapeutic and Cytostatic Drugs

Theranostic inorganic-organic hybrid nanoparticles (IOH-NPs) with a cocktail of chemotherapeutic and cytostatic drugs and a composition Gd23+[(PMX)0.5(EMP)0.5]32-, [Gd(OH)]2+[(PMX)0.74(AlPCS4)0.13]2-, or [Gd(OH)]2+[(PMX)0.70(TPPS4)0.15]2- (PMX: pemetrexed, EMP: estramustine phosphate, AlPCS4: aluminum(III) chlorido phthalocyanine tetrasulfonate, TPPS4: tetraphenylporphine sulfonate) are presented for the first time. These IOH-NPs are prepared in water (40-60 nm in size) and have a non-complex composition with outstanding drug loading (71-82% of total nanoparticle mass) of at least two chemotherapeutic or a mixture of cytostatic and photosensitizing agents. All IOH-NPs show red to deep-red emission (650-800 nm) to enable optical imaging. The superior performance of the IOH-NPs with a chemotherapeutic/cytostatic cocktail is validated based on cell-viability assays and angiogenesis studies with human umbilical vein endothelial cells (HUVEC). The synergistic anti-cancer effect of the IOH-NPs with a chemotherapeutic cocktail is shown in a murine breast-cancer cell line (pH8N8) and a human pancreatic cancer cell line (AsPC1), whereas the synergistic cytotoxic and phototoxic efficacy is verified in response to illumination of HeLa-GFP cancer cells, MTT assays with human colon cancer cells (HCT116), and normal human dermal fibroblasts (NHDF). HepG2 spheroids as 3D cell cultures prove the effective uptake of the IOH-NPs with high uniform distribution and the release of the chemotherapeutic drugs with the strong synergistic effect of the cocktail of drugs.


Analytical Equipment
Scanning electron microscopy (SEM) was carried out with a Zeiss Supra 40 VP (Zeiss, Germany), equipped with a field emission gun (acceleration voltage 5 kV, working distance 3 mm).Samples were prepared by spraying a diluted aqueous suspension of the as-prepared IOH-NPs with a mist maker on a silica wafer that was left for drying overnight.
Energy-dispersive X-ray spectroscopy (EDXS) was performed with an Ametek EDAX device (Ametek, USA) mounted on the above described Zeiss SEM Supra 40 VP scanning electron microscope.For the analysis, the IOH-NPs were dried at 50°C and thereafter pressed to dense pellets in order to guarantee for a smooth surface and a quasi-infinite layer thickness.
These pellets were fixed with conductive carbon pads on aluminium sample holders.An acceleration voltage of 30 kV was used for these measurements.
Dynamic light scattering (DLS) and zeta potential measurements were carried out on a Zetasizer Nano-ZS (Malvern, United Kingdom) with a 633 nm laser and backscattering geometry (173°).For DLS measurements, the aqueous IOH-NP suspensions were diluted 1:10 or 1:20 with demineralized water.The zeta potential of the as-prepared nanoparticles was also measured using these diluted aqueous suspensions.
Fourier-transform infrared (FT-IR) spectra were recorded on a Bruker Vertex 70 FT-IR spectrometer (Bruker, Germany) in the range from 4000 to 400 cm -1 with a resolution of 4 cm -1 .
For this purpose, 1 mg of dried IOH-NPs was pestled with 300 mg of KBr and pressed to a pellet, which thereafter was measured in transmission.
Differential thermal analysis/thermogravimetry (DTA/TG) was performed with a STA409C device (Netzsch, Germany).The measurements were performed in air to guarantee for total combustion of the organic content.The IOH-NPs (20 mg in corundum crucibles), pre-dried at 100 °C for 5 h, were heated to 1200 °C with a rate of 5 K/min.X-ray diffraction (XRD) measurements of the as-prepared IOH-NPs as well as of the residue after total organic combustion of these IOH-NPs (TG analysis) were performed with a Stadi MP diffractometer (STOE & Cie, Germany) using a Cu-K α1 radiation source (λ = 154.05pm) and a germanium-(111)-monochromator.

Elemental analysis (EA, C/H/N/S analysis) was performed via thermal combustion with an
Elementar Vario Microcube device (Elementar, Germany) at a temperature of 1150 °C.The samples were pre-dried at 100°C for 5 h to remove the remaining solvent.
Photoluminescence (PL) measurements were performed with a Horiba Jobin Yvon Spex Fluorolog 3 (Horiba Jobin Yvon, France) equipped with a 450 W Xe-lamp and double-grating excitation and emission monochromators.
Particle size and colloidal properties were characterized based on scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta-potential analysis (see main paper Table 1, Figure 2; Figures S1-S5).Thus, the IOH-NPs exhibit particle diameters of 40 to 60 nm according to SEM and hydrodynamic diameters of 60 to 100 nm according to DLS.Zetapotential measurements shown negative charging of -15 to -35 mV.According to x-ray diffraction (XRD), the IOH-NPs are non-crystalline (Figure S4).Fourier-transform infrared (FT-IR) spectroscopy evidences the presence of the respective cytostatic anion (see main paper Total organics combustion via thermogravimetry (TG) and elemental analysis (EA) confirm the chemical composition of the IOH-NPs (see main paper Table 2; Figures S1-S5).Finally, the thermal remnants of the TG analyses were analysed by XRD, resulting in Gd 2 O 3 , GdPO 4 , and Gd 3 PO 7 as residual phases (Figure S5).After correcting the experimental data for a release of 5.0 wt-% of adsorbed H 2 O (Table S1, Figure S1), the thermal decomposition of For [Gd(OH)] 2+ [EMP] 2-the experimental data were also corrected for 5.0 wt-% of adsorbed water (Table S1, Figure S2) and relate to a thermal decomposition according to: 2      ICG is clinically approved and known for deep-red emission, which, however, is weak for freely dissolved ICG.S1 Due to the great number of ICG anions in a single IOH-NP, however, the deep-red emission is here sufficient for fluorescence detection.On the other hand, the ICG load is nevertheless low (about 5-6 mol-%) in comparison to the drug load, so that the drug load per nanoparticle is reduced only slightly.This is expressed by the chemical formula
DUT shows intense deep-red emission and is required only with very small amounts ( 1

In vitro Studies for IOH-NPs with Chemotherapeutic Cocktail
Cell culture.The adherent human pancreatic cancer cell line AsPC-1 was purchased from ATCC (Rockville, USA).The adherent murine breast cancer cell line pH8N8 was maintained and cultured as described before.S2 Cells were cultivated at 37 °C in a humidified atmosphere of 5% CO 2 .AsPC1 cells were grown in RPMI-1640 media (Life Technologies, Germany) and pH8N8 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies, Germany), both supplemented with 10% fetal bovine serum (FBS Gold, PAA Laboratories Gold).
Confocal fluorescence microscopy.To study IOH-NP uptake, pH8N8 or AsPC1cells were plated in a concentration of 50.000 cells per well on poly-L-lysine-coated glass-cover slips and allowed to attach for two days.Afterwards, the cell-culture medium was replaced by a fresh medium supplemented with 50 µL/mL of Gd 2 3+ [(PMX) 0.50 (EMP) 0.49 (DUT) 0.01 ] 3 2-IOH-NPs.
The ICG-derived fluorescence was recorded using a high sensitivity ORCA-AG digital camera (Hamamatsu, Japan) and the 708/75 nm bandpass filter for the excitation and 809/81 nm bandpass filter for the emission.DAPI was excited at 365/25 nm.The emission (blue) was collected at 445/50 nm.The DUT-derived fluorescence was visualized using an SP5 confocal microscope (Leica, Germany).DAPI was excited with a 405 nm laser, and the emission was collected at 415-500 nm.DUT was excited using a 633 nm laser, and the emission was collected at 645-780 nm.Image generation and processing were performed with the AxioVision Rel.4.6 software (Zeiss, Germany) and FIJI S3 (National Institutes of Health, USA) (Figure S11; see main paper: Figure 3).AsPC1 and pH8N8 cells were plated in a 96-well plate in a concentration of 10.000 cells per well in the corresponding cell-culture medium and allowed to attach overnight.On the next day, the cell-culture medium was replaced by 200 µL of fresh medium (green pillars) or medium supplemented with increasing amounts (1, 5, 10, 50 µL) of Gd Figure 4).The metabolic activity was assessed either directly after adding the IOH-NPs or dissolved drugs (0 h) as well as after 24 and 72 h of treatment using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega).The absorbance of the metabolised substrate was measured at 490 nm.The experiment was performed in triplicate.Additionally, the absorbance of each substance diluted in the medium (IOH-NPs or dissolved drugs) was measured in cell-free conditions (black pillars: Figures S12,S13; see main paper: Figure 4).The cytotoxic efficacy of IOH-NPs with chemotherapeutic cocktail, first of all, was evaluated in in vitro studies with the single-drug Gd 3+ 2 [PMX] 2- 3 and [Gd(OH)] 2+ [EMP] 2-IOH-NPs using pH8N8 murine breast cancer cells and AsPC1 human pancreatic cancer cells.On the one hand, these results were compared with dual-drug Gd 3+ 2 [(PMX) 0.5 (EMP) 0.5 ] 2- 3 IOH-NPs (see main paper: Figure 4).On the other hand, the activity of the IOH-NPs was compared with the freely dissolved drugs PMX (Na 2 (PMX)×7H 2 O) (Figure S12), EMP (Na 2 (EMP) (Figure S13), as well as untreated cells (phosphate-buffered saline/PBS only) (Figures S12,S13; see main paper: Figure 4).The concentrations of the freely dissolved drugs were according to their dose in the IOH-NPs.Dotted red lines indicate the self-absorption of medium, drugs, and/or IOH-NPs, which needs to be taken into account to evaluate the cell growth.
In addition to the aforementioned cytotoxic efficacy (Figures S12,S13; see main paper:

Material Characterization of IOH-NPs with Phototoxic Agents
Prior to in vitro studies for IOH-NPs with cytotoxic and phototoxic agents, the formation of reactive oxygen species (ROS) was proven with single-agent Gd 4 3+ [AlPCS 4 ] 3 4-and La 4 3+ [TPPS 4 ] 3 4-IOH-NPs.These studies were performed and published before.S4 Therefore, only the essential aspects are summarized here as far as they are relevant for the novel dualfunction IOH-NPs, which we report for the first time.
For both IOH-NPs, ROS formation as well as the quantum yield (φ Δ ) for singlet oxygen production were evaluated using different methods.For Gd 4 3+ [AlPCS 4 ] 3 4-, φ Δ was determined by a relative method using DPBF (1,3-diphenylisobenzofuran) as a chemical quencher for 1 O 2 oxygen.S4,S5 Accordingly, φ Δ is proportional to the decrease of the DBPF absorption band under illumination and can be recorded via UV-Vis spectroscopy along with the irradiation time (Figure S22a).In difference, φ Δ for La 4 3+ [TPPS 4 ] 3 4-IOH-NPs was determined by the iodide method based on the reaction of 1 O 2 with I -in the presence of (NH 4 ) 2 MoO 4 as a catalyst.S4,S5 As a result, I 3 -is produced in an amount directly proportional to the generated 1 O 2 .Here, the increase of the absorption band of I 3 -can be monitored spectroscopically along with radiation time (Figure S22b).It needs to be noticed that the more common DPBF method cannot be used in the case of La 4 3+ [TPPS 4 ] 3 4-since its absorption overlays the DPBF band.S4,S5 Based on the above described methods, the quantum yield for 1 O 2 production was determined to 37% for Gd 4 3+ [AlPCS 4 ] 3 4-and 49% for La 4 3+ [TPPS 4 ] 3 4-.S4 These values are similar to freely dissolved H 4 (AlPCS 4 ) (34%) and H 4 (TPPS 4 ) (51%) in aqueous solution.S5    (2.6, 13, 26 µg/mL) for 48 h.The concentrations of the freely dissolved agents were according to their dose in the IOH-NPs.Eventually, the cells were illuminated for 30 min at 700 nm after 24 h and further incubated for a total of 48 h.The experiments were performed in triplicates.
Values are expressed as the mean ±SD (n = 3).Angiogenesis.To determine the impact of the IOH-NPs on the formation of new microcapillaries from endothelial cells, 8-well μ-slides were coated with 60 µL Geltrex ® solution (Thermo Fischer Scientific, Germany) per well and incubated overnight.4×10 4 HUVEC in 160 µL EGM-2 medium were seeded in each well.The IOH-NPs and the freely dissolved photosensitizers were added.After 1 h as well as after 3 h, illuminations with 700 nm light for AlPCS 4 (40 min) and white light for TPPS 4 (3 min) were performed, respectively, followed by further incubation of 24 h.Thereafter, the cell samples were stained with Hoechst 33342 (2.0 mg/mL) and examined via confocal microscopy (TCS SPE DMI4000B inverted microscope, Leica Microsystems, Germany).The images were taken at excitation/emission: 405 nm/410-450 nm for Hoechst 33342.
3D cell culture.Spheroids were used for drug efficacy testing for the detection of cytotoxic, phototoxic and anti-proliferative effects in 3D cell culture (Figures S26,S27; see main paper: Figure 11).Cells were seeded at a concentration of 3000 cells per well in agarose-treated 96well plates, and cultured for 3 days before being treated with the IOH-NPs or the freely dissolved agents.One day after treatment, the cells were eventually illuminated for 30 min at 700 nm (for AlPCS 4 ) or white light (for TPPS 4 ) and further incubated.Toxicity assays were performed as previously described.To examine the spheroid growth over time, spheroids were imaged 7 days using light microscopy.The spheroid diameter was measured by LAS X Leica Software.Values are expressed as the mean ±SD (n = 6).

Figure S1 .
Figure S1.Particle characterization and chemical composition of Gd 3+ 2 [PMX] 2- 3 IOH-NPs: a) Scheme of synthesis with structure of anion, b) Particle size and shape according to SEM, c) Particle size distribution according to DLS and SEM, d) Zeta potential of aqueous suspension, e) FT-IR spectrum with pure PMX as a reference, f) TG analysis.

Figure S2 .
Figure S2.Particle characterization and chemical composition of Gd 3+ 2 [EMP] 2- 3 IOH-NPs: a) Scheme of synthesis with structure of anion, b) Particle size and shape according to SEM, c) Particle size distribution according to DLS and SEM, d) Zeta potential of aqueous suspension, e) FT-IR spectrum with pure EMP as a reference, f) TG analysis.

Figure S3 .
Figure S3.Particle characterization and chemical composition of Gd 3+ 2 [(PMX) 0.5 (EMP) 0.5 ] 2- 3 IOH-NPs: a) Scheme of synthesis with structure of anion, b) Particle size and shape according to SEM, c) Particle size distribution according to DLS and SEM, d) Zeta potential of aqueous suspension, e) FT-IR spectrum with pure PMX and EMP as references, f) TG analysis.
Figures S7,S8).Again, the IOH-NPs exhibit particle diameters of 40 to 60 nm (SEM) and hydrodynamic diameters of 60 to 100 nm (DLS) and negative charging of -15 to -35 mV.FT-IR spectra evidence the presence of PMX, AlPCS 4 or TPPS 4 (see main paper Figure2;Figures S7,S8).The characteristic vibrations of the cytostatic and the photosensitizing anions are observed and well in agreement with the starting materials as references (PMX: v(C-H): Figure S7.Particle characterization and chemical composition of [Gd(OH)] 2+ [(PMX) 0.74 (AlPCS 4 ) 0.13 ] 2-IOH-NPs: a) Scheme of synthesis with structure of anion, b) Particle size and shape according to SEM, c) Particle size distribution according to DLS and SEM, d) Zeta potential of aqueous suspension, e) FT-IR spectrum with pure PMX and AlPCS 4 as references, f) TG analysis.
Figure S8.Particle characterization and chemical composition of [Gd(OH)] 2+ [(PMX) 0.70 (TPPS 4 ) 0.15 ] 2-IOH-NPs: a) Scheme of synthesis with structure of anion, b) Particle size and shape according to SEM, c) Particle size distribution according to DLS and SEM, d) Zeta potential of aqueous suspension, e) FT-IR spectrum with pure PMX and TPPS 4 as references, f) TG analysis.

Figure S12 .
Figure S12.In vitro studies of IOH-NPs with single drug and chemotherapeutic cocktail on AsPC1 cells.MTT-based viability test after 0 to 72 h of treatment with (a) untreated cells, (b) treated with the indicated IOH-NP concentration (1-50 µL/200 µL), and (c) treated with the freely dissolved drugs.The concentrations of the freely dissolved drugs were according to their dose in the IOH-NPs.Dotted red lines indicate the self-absorption of medium, cells, and/or drugs.Error bars correspond to standard error of n = 4.

Figure S13 .
Figure S13.In vitro studies of IOH-NPs with single drug and chemotherapeutic cocktail on pH8N8 cells.MTT-based viability test after 0 to 72 h of treatment with (a) untreated cells, (b) treated with the indicated IOH-NP concentration (1-50 µL/200 µL), and (c) treated with the freely dissolved drugs.The concentrations of the freely dissolved drugs were according to their dose in the IOH-NPs.Dotted red lines indicate the self-absorption of medium, cells, and/or drugs.Error bars correspond to standard error of n = 4.

Figure 4 )
Figure 4), cell-based assays monitored by Incucyte were performed over 0-72 hours with AsPC1 and pH8N8 cells incubated with a concentration of NPs of 1-50 µL/200 L medium (Figures S14-S21).All IOH-NPs show a clear time-dependent and concentration-dependent cytotoxic effect on the tumour cells.Even at low concentration (5 µL/200 L) and short time of incubation (24 hours), the cell morphology is changing significantly.Detachment and swelling of cells, beginning deformation of nuclei, and accumulation of perinuclear vesicular structures indicate beginning cell death.This confirms the chemotherapeutic IOH-NPs not only to be effectively internalized by the cells but to also evidently release their drug load, resulting in the expected concentration-dependent cytotoxic efficacy.Similar to the results of the MTTbased viability test, pH8N8 cells are more affected by EMP (Figures S18-S21), whereas PMX shows a higher activity on AsPC1 cells (Figures S14-S17).Beside cell remains, an increasing number of IOH-NP agglomerates is visible, especially at high IOH-NP concentrations (10 and 50 µL/200 L).At high IOH-NP concentrations, thus, IOH-NPs are still present without having released their full drug load although all cells are dead.Beside this qualitative evaluation, a quantification of cell density reflecting cell growth is difficult since the remains of dead cells and IOH-NP agglomerates are difficult to differentiate (Figures S14-S21).

Figure S24 .
Figure S24.Cell viability assays for PMX on human umbilical vein endothelial cells (HUVEC) ±illumination.HUVEC cells were treated with different concentrations of PMX (2.2, 11,22 μg/mL) (corresponding to the PMX concentration in the 5, 25, 50 µg/mL IOH-NPs).24 h after treatment, the samples were exposed to an illumination at 700 nm for 30 min.

Table S1 .
Data of the thermogravimetric analysis with correction of the experimental data for the amount of absorbed water.

;
Figures S7,S8).The thermal remnants of the TG analyses were analysed by XRD, resulting in Gd 2 O 3 and Gd 4 Al 2 O 9

Table S2 .
Data of the thermogravimetric analysis with correction of the experimental data for the amount of absorbed water.