Cu-HKUST-1 and Hydroxyapatite–The Interface of Two Worlds toward the Design of Functional Materials Dedicated to Bone Tissue Regeneration

A novel composite based on biocompatible hydroxyapatite (HA) nanoparticles and Cu-HKUST-1 (Cu-HKUST-1@HA) has been prepared following a layer-by-layer strategy. Cu-HKUST-1 was carefully selected from a group of four Cu-based metal–organic frameworks as the material with the most promising antimicrobial activity. The formation of a colloidal Cu-HKUST-1 layer on HA nanoparticles was confirmed by various techniques, e.g., infrared spectroscopy, powder X-ray diffraction, N2 sorption, transmission electron microscopy imaging, electron paramagnetic resonance, and X-ray absorption spectroscopy. Importantly, such a Cu-HKUST-1 layer significantly improved the nanomechanical properties of the composite, with Young’s modulus equal to that of human cortical bone (13.76 GPa). At the same time, Cu-HKUST-1@HA has maintained the negative zeta potential (−16.3 mV in pH 7.4) and revealed biocompatibility toward human dermal fibroblasts up to a concentration of 1000 μg/mL, without inducing ex vivo hemolysis. Chemical stability studies of the composite over 21 days in a buffer-simulated physiological fluid allowed a detailed understanding of the transformations that the Cu-HKUST-1@HA undergoes over time. Finally, it has been confirmed that the Cu-HKUST-1 layer provides antibacterial properties to HA, and the synergism reached in this way makes it promising for bone tissue regeneration.


General methods
All solid products were analyzed by powder X-ray diffraction (PXRD) using a X'Pert PRO diffractometer (PANalytical) with Cu Kα radiation (λ = 1.5406 Å). The mid-infrared spectra (IR) were measured using the Nicolet iSTM50 FT-IR spectrometer. The spectra were recorded for the KBr pellets. The micro-Raman apparatus (inVia™ Renishaw) was used to register the Raman spectra with a 514 nm excitation line. Thermogravimetric analysis (TGA) was performed using a Setaram SETSYS TG-DTA 16/18 at a heating rate of 10 ºC/min in flowing air. The specific surface area was determined based on N2 sorption measurements. The N2 adsorption-desorption isotherms were obtained at 77 K using a Micromeritics 3Flex Surface Characterization Analyzer. Prior to isotherm acquisition, the materials were activated and outgassed (150 ºC, 1.3 kPa) for 12 h. MicroActive software was used to determine specific surface area according to the Brunauer-Emmett-Teller (BET) method. HR-TEM images with Fast Fourier Transform (FFT) and STEM-EDS elemental maps were performed using a FEI Titan G2 60-300. The particle size of HA was calculated from the size of 100 particles determined by ES Vision software using TEM images. The powders and filtrates composition were with Zetasizer Nano ZS (Malvern Instruments) for HA and Cu-HKUST-1@HA (1.5 mg/mL in DPBS or distilled water) sonicated for 5 min before measurement. The potential was measured six times each in two independent experiments (each measurement being the average of 100 runs) both in DPBS and H2O. The mean values and standard deviations were equal to -16.3 ± 1.7 mV for Cu-HKUST-1@HA, -14.6 ± 3.7 mV for HA in DPBS, and -9.1 ± 0.7 mV for Cu-HKUST-1@HA and -14.2 ± 2.3 mV for HA in H2O. The EPR spectra were measured at 77 K using a Bruker Elexsys 500 CW-EPR spectrometer operating at the X-band frequency (~9.7 GHz), equipped with frequency counter (E 41 FC) and NMR teslameter (ER 036TM). The spectra were measured with a modulation frequency of 100 kHz, microwave power of 10 mW, modulation amplitude of 10 G, time constant of 40 ms and a conversion time of 160 ms. The first derivative of the absorption power was recorded as a function of the magnetic field value. The experimental spectra were simulated using the computer program DoubletExact (S = 1/2), written by Prof. Andrew Ozarowski from NHMFL, Florida State University.

Synthesis of Cu-HKUST-1
Cu-HKUST-1 was prepared by a liquid-assisted grinding (LAG) method previously described by Steenhaut et al. with some modifications. 2 Typically, Cu(OAc)2·H2O (0.040 g, 0.2 mmol) and H3btc (0.028 g, 0.13 mmol) were pre-ground in an agate mortar and then introduced into a milling tube together with 0.5 mL of ethanol. The mixture was subjected to milling at 6000 rpm for 30 min in the homogenizer (IKA ULTRA-TURRAX® Tube Drive Disperser). Milling was carried out in an appropriate tube of 15 mL containing ten stainless-steel balls of 5 mm. Afterwards, the resulting precipitate was washed twice with ethanol (99.8%) (2 × 10 mL) and dried in vacuo.
The mixture was left overnight at reflux under stirring. The formation of a brown suspension was observed, while a violet precipitate was finally formed after cooling to room temperature. The resulting precipitate was filtered off, washed with distilled water (2 × 10 mL), and dried in vacuo.

Synthesis of hydroxyapatite (HA)
HA nanoparticles (Ca10(PO4)6(OH)2) with rod-like shapes were synthesized according to a previous method. 6 Briefly, H3PO4 (0.15 M) was slowly dropped to an aqueous suspension of Ca(OH)2 (ca. 0.17 M), resulting in the precipitation of HA. The reaction mixture was further stirred at 37 °C for 24 h and left standing for the next 24 h. Afterwards, the precipitate was separated by centrifugation, washed with water (3 × 20 mL), and dried at 60 o C overnight. Then, to obtain the rod-like shape, the HA powder was hydrothermally treated (at 200 o C for 24 h). After cooling to room temperature, the HA powder was washed with distilled water (2 × 20 mL) and dried at 60 o C overnight.

Synthesis of Cu-HKUST-1@HA
The new Cu-HKUST-1@HA composite was prepared following a layer-by-layer method carried out at room temperature. For this purpose, HA (0.5 g) was suspended in an ethanolic solution of H3btc (10 mL, 6.66 mM) and the mixture was stirred for 1 h. Afterwards, the solid was centrifuged and washed with ethanol (1 × 10 mL). The resulting material was then soaked in an ethanolic solution of Cu(OAc)2·H2O (10 mL, 10 mM) for 1 h. Similarly, the resulting product was centrifuged and washed with ethanol (1 × 10 mL). This procedure, which represents one cycle of the layer-by-layer process, was repeated 10 times. Finally, the composite was dried at 60 o C overnight.
Neglecting the presence of Cu2O, the chemical composition of the composite was calculated as

Nanomechanical properties
In order to determine the nanomechanical properties, the test materials were applied to a polished Ti13Zr13Nb titanium alloy surface with a roughness of 0.13 µm. To 0.1 g of each material, 1 mL of ethanol (99.8%, Sigma Aldrich) was added, then pipetted and subjected to shaking using a laboratory shaker (1500 rpm for 20 s). The colloidal suspensions thus prepared were applied to the titanium surfaces and allowed to evaporate.
To determine the nanomechanical properties, a nanoindenter (NanoTest Vantage, MicroMaterials, The United Kingdom) with a Berkovich diamond indenter was used. Ten independent measurements were performed on each sample with a maximum force of 1000 µN in loading time 40 s, hold with a maximum force of 5 s, and unloading time of 30 s. The distance between indents was 20 µm. After each indentation test, temperature drift was allowed for 10 s. The nanohardness reduced Young's modulus, plastic work, and elastic work using Oliver-Pharr methods were determined. To obtain Young`s modulus from the reduced Young's modulus values, the Poisson's ratio for the tested materials was assumed to equal 0.28.

In vitro cytotoxicity assay
The in vitro cytotoxicity was assessed by using a human dermal fibroblast (HDF) cell line. HDF cells Subsequently, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test based on the ability to reduce MTT by mitochondrial dehydrogenases was performed in triplicate to assess the cell metabolic activity (viability).

Hemolytic activity
The hemolytic activity of Cu-HKUST-1@HA was determined based on the procedure reported by  Figure S1. Powder X-ray diffraction patterns (a) and IR spectrum (b) of Cu-HKUST-1.   Binding Energy (eV) Figure S8. The high-resolution Cu 2p XPS spectra of Cu-HKUST-1@HA. Binding Energy (eV) Figure S9. The high-resolution Ca 2p XPS spectra of Cu-HKUST-1@HA. S13 Binding Energy (eV) Figure S10. The high-resolution P 2p XPS spectra of Cu-HKUST-1@HA.