Introduction

Nanoparticle therapeutics are now a rapidly growing and considerably promising treatment modality for cancer. The vast majority of these nanoparticles are used as carriers modified with numerous targeting ligands for cancer chemotherapeutics in order to improve the drug efficacy and reduce the side effects (Peer et al. 2007; Misra et al. 2010). By contrast, hydroxyapatite nanoparticles (HAPNs) without any surface modifications have shown some activity in selectively killing cancer cells, indicating more simple and controllable preparation as well as fewer additional ingredients to obtain a potential anticancer medicine. Moreover, as the major inorganic constituent of the hard tissue of humans and animals, HAPNs possess excellent biocompatibility and bioactivity. Previously, we prepared rod-like hydroxyapatite nanoparticles by wet precipitation method, which showed cancer-specific cytotoxicity (Tang et al. 2014). Our recent work further proved that HAPNs could selectively inhibit the growth of lung cancer cells in vitro and in vivo because of enhanced internalization, mitochondrial targeting, and sustained calcium elevation in cancer cells (Sun et al. 2016). In order to promote the application of HAPNs in cancer therapy, it is necessary to understand the role of serum in biological environments on altering the nanoparticle dispersion state and anticancer effect (Shannahan 2017; Tedja et al. 2012). However, to date, little attention has been paid to understanding the interaction between nanoparticles and fetal bovine serum (FBS). There is a need to detect and confirm the adsorption of proteins in FBS on HAPN surface and clarify the effect of existence of proteins on HAPN aggregation and toxicity on cancer cells.

Despite the growing interest and remarkable speed of development in nanomedicine, nowadays the large global investments in cancer nanomedicine have not been translated into significant clinical use as expected, which may partly result from an incomplete understanding of nano-bio interactions, such as protein adsorption and nanoparticle agglomeration in biological conditions (Venditto and Szoka Jr 2013; Charbgoo et al. 2018; Shi et al. 2017; Albanese and Chan 2011). Fetal bovine serum containing numerous proteins is commonly used in cell culture studies for cell growth and proliferation. It is known that once nanoparticles are transferred into cell culture medium containing FBS, proteins will be adsorbed onto nanoparticle surface forming so-called protein corona and affect the features and properties (for example, stability, particle agglomerate size, or dissolution) of nanoparticles (Pyrgiotakis et al. 2013; Shannahan 2017; Foroozandeh and Aziz 2015). The term “protein corona” was first raised to define the protein coating on nanoparticle surfaces in 2007 (Cedervall et al. 2007). The protein corona is usually composed of an inner layer called the hard corona which has a long lifetime and an outer layer called the soft corona which keeps exchanging the proteins (Mahmoudi et al. 2011). Additionally, the composition of protein layers mainly depends on the surface properties of nanoparticles, potentially altering the properties of nanoparticles (Mortensen et al. 2013; Nguyen and Lee 2017). There have been several studies about the effect of protein adsorption on the stability and dispersion of nanoparticles in biological systems. The size of magnetic nanoaggregates can be maintained below 200 nm because of the stabilization via adsorption with FBS proteins (Wiogo et al. 2011). FBS weakened agglomeration and sedimentation properties of cationic zinc oxide nanoparticles (Anders et al. 2015).

Apart from the influence of proteins in cell culture conditions on nanoparticle dispersion, it is critical to understand the role of fetal bovine serum in cell response of nanoparticles, leading to an accurate interpretation of in vitro results. Cell responses like cellular internalization and nanotoxicity will be highly dependent on the protein corona and the agglomeration state of nanoparticles (Behzadi et al. 2017; Maiorano et al. 2010; Bruinink et al. 2015). In terms of the cells, they can first recognize proteins, which cover the surface of nanoparticles (Lynch et al. 2009). The proteins will impart biological identity to nanoparticles and mediate nanoparticle dispersion state, which jointly alter the internalization and cytotoxicity of nanoparticles. Both gold and silver nanoparticles were exposed to human serum albumin and immunoglobulin, leading to good dispersion and higher cellular uptake, while fibrinogen resulted in severe agglomeration and relatively less cell internalization (Sasidharan et al. 2015). The cytotoxicity of silver nanoparticles was enhanced in contact with serum proteins (Barbalinardo et al. 2018). The presence of serum protein can avoid silica nanoparticle aggregation and provide nanoparticles favorable interactions with cells (Catalano et al. 2015). The agglomerate size of hydroxyapatite nanoparticles was reduced after coating with citrate or dispersing agent, which subsequently governed cytotoxicity, particle uptake, and the formation of surface-connected compartment (SCC) in human macrophages (Mueller et al. 2014).

In this work, we aimed to explore the influence of fetal bovine serum on HAPN dispersion in cell culture environment and the subsequent impact on nanoparticle–cell interaction. HAPNs mixed with different concentrations of FBS were prepared and denoted as HAPN–FBS complex. Transmission electron microscope (TEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, and thermogravimetric analysis (TG) were performed here to demonstrate the formation of protein coronas on the surface of nanoparticles. Real-time adsorption of FBS on the surface of HAPNs was measured by quartz crystal microbalance with dissipation (QCM-D) technique. The change of agglomeration state of HAPNs after being incubated with FBS was recorded by dynamic light scattering (DLS), inverted microscope, and scanning electron microscope (SEM). The stability of HAPN–FBS complex in solution was also analyzed by DLS. Here, we chose MG-63 cells from human osteosarcoma as a model to investigate the cell response to HAPN–FBS complex, as well as the effect of HAPNs on bone-related cancer since HAPNs have been frequently utilized for bone tissue engineering (Zhou and Lee 2011). The internalization and toxicity of HAPN–FBS complex on MG-63 cells were analyzed. This work provided a deep insight into how FBS affects the HAPN agglomeration and subsequent cell interaction.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), FBS, and phosphate buffer solution (PBS, pH = 7.4) were from Gibco Life Technologies (CA, USA). MG-63 cells were obtained from the American Type Culture Collection (ATCC, VA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), 3-aminopropyltriethoxysilane (APTES), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (MO, USA). Hoechst 33258 was from Biotium (CA, USA).

Preparation of HAPNs and HAPN–FBS complex

HAPNs were synthesized using an aqueous precipitation method by mixing 0.2 M Ca(NO3)2 solution and 0.2 M (NH4)2HPO4 solution under pH 10 at 10 °C for 30 h. Thereafter, nanoparticles were collected at a speed of 12,000 rpm for 15 min and washed several times with deionized (DI) water and anhydrous ethanol. After freeze-drying for 24 h, nanoparticles were calcinated at 600 °C for 2 h and kept at room temperature for further use.

A total of 5 mg HAPNs were added into 5 mL DMEM solution containing 0, 2, 5, 8, and 10% FBS. The suspensions were sonicated for 10 min and then stirred continuously for 4 h to prepare the dispersions of HAPN–FBS complex.

TEM images of HAPN–FBS complex

In order to clearly observe the surface of the particles, 100 μL of HAPN–FBS (0, 5, and 10%) complex dispersions were deposited onto carbon-coated copper grids and allowed to dry overnight. Samples were viewed with a Philips CM12 TEM (Philips Electronic Instruments, USA) with an AMT XR111 digital camera operating at 80 kV.

ATR-FTIR spectrum of HAPNs and HAPN–FBS complex

HAPN–FBS (0, 2, 5, 8, and 10%) complexes were washed three times by DI water and collected by centrifuging at 12,000 rpm for 15 min. The samples were freeze-dried on a lyophilizer for 24 h. The dry powder of HAPNs and HAPN–FBS complex was examined by ATR-FTIR spectroscopy (Nicolet iS10, Thermo Fisher Scientific, USA) to confirm if serum proteins have been attached on the surface of the HAPNs.

Real-time FBS protein adsorption on HAPN surface

QCM-D (Q-Sense E4, Q-Sense AB, Sweden) was performed to measure the adsorption of FBS on HAPNs in situ. QCM-D sensors were sonicated in tetrahydrofuran and rinsed with DI water. Afterwards, the sensors were immersed in a 5:1:1 mixture of Milli-Q water, hydrogen peroxide, and ammonia, which was heated to 75 °C. Then, 10 min later, the sensors were rinsed with DI water, dried with N2 gas, and treated under UV for 20 min.

An electrophoretic deposition method was applied to deposit HAPNs onto QCM-D sensors (Huang et al. 2015). The sensor was installed as the negative pole and the platinum electrode as the positive pole. HAPN suspension in ethanol at 1 wt% was used as electrolyte. Voltage at 100 V/cm DC was operated for 5 min to coat the sensor with HAPNs on the surface. The sensors were subsequently sonicated in ethanol for 1 min to remove the remaining unabsorbed HAPNs.

The prepared QCM-D sensors with HAPN layer were mounted in the QCM-D cell. DMEM and DMEM with 10% FBS solutions were injected into the cell after the frequency was stabilized in PBS (pH = 7.4). The frequency shifts (∆F) were monitored and recorded to observe the FBS adsorption onto HAPN surface.

The size distribution of HAPN–FBS complex

The dispersions of HAPN–FBS (0, 2, 5, 8, and 10%) complexes (100 μL) were added in 900 μL of PBS for DLS detection to reduce the detection error from high particle concentration and color of phenol red in DMEM. The hydrodynamic size of HAPN–FBS complex was measured by DLS (DelsaNano S, Beckman Coulter, USA). DelsaNano S can measure the particle size ranging from 0.6 nm to 7 μm. The particle size was calculated using the Stokes–Einstein equation.

Microscopy images of HAPN–FBS complex

Observation under a microscope is a direct method to visualize the variation of nanoparticle dispersion. A total of 200 μL of HAPN suspensions (1000 μg/mL) after stirring in DMEM with different concentrations of FBS (0, 2, 5, 8, and 10%) for 4 h were transferred to a 96-well plate. After the particles precipitated to the bottom of the plate over 2 h, the agglomeration state of HAPN–FBS complex was observed under an inverted Zeiss AX10 microscope (Carl Zeiss AG, Germany).

SEM images of HAPN–FBS complex

A filtration method was employed to reveal the state of HAPN–FBS complex in cell culture medium (Mueller et al. 2014). HAPN–FBS (0, 2, 5, 8, and 10%) complexes suspensions were diluted to 125 μg/mL with DMEM in the absence or presence of 2, 5, 8, and 10% FBS. A total of 1 μm membrane filters (13 mm ø Nuclepore Track Etch Membranes, Whatman) were fixed by filter holders (13 mm ø Pop Top membrane holders, Whatman). Afterwards, 3 mL of particle suspensions were filtered through the membranes using syringes. Membranes were rinsed with 2 mL of DI water to remove medium components and subsequently dried at 60 °C for 2 h. Then, membranes were pressed onto sticky carbon pads attached to aluminum stubs. Samples were viewed by SEM (LEO 1550, Carl Zeiss AG, Germany) to examine the remaining particles on the membranes.

The stability of HAPN–FBS complex in a short time period

To study the effect of FBS on the sedimentation and re-agglomeration of HAPNs, HAPN–FBS (0, 2, 5, 8, and 10%) complexes were transferred into disposable cuvettes and kept still in room temperature after being diluted 1:10 in PBS. Size measurement was performed by DLS after 0, 30, 60, and 90 min to detect the variation in size distribution of HAPN–FBS complex.

The stability of HAPN–FBS (10%) complex over 5 days

The size distribution of HAPN–FBS (10%) complex within 5 days was measured to evaluate the long-term stability. HAPNs (1000 μg/mL) were stirred for 4 h in DMEM with 10% FBS and then preserved at 37 °C in 5% CO2 for 0, 1, 2, and 5 days. At each time point, HAPN–FBS (10%) complex was diluted to 100 μg/mL HAPNs with PBS and measured by DLS.

Cellular uptake detected by laser scanning confocal microscope

FITC-labeled HAPNs were prepared to visualize the nanoparticles inside the cells. APTES (1 mL) was reacted with 0.05 g of HAPNs in 50 mL of anhydrous ethanol by stirring at 74 °C for 3 h. Then, 0.025 g of FITC was added into the reaction and stirred for 6 h in dark place. The synthesized materials were washed several times with anhydrous ethanol and DI water alternately until no free FITC remained. FITC-labeled nanoparticles (FITC-HAPNs) were then freeze-dried for 24 h.

MG-63 cells were seeded into a 6-well plate with a glass bottom and cultured overnight at 37 °C with 5% CO2. FITC-HAPNs (1000 μg/mL) were stirred in DMEM with 0, 5, and 10% FBS, respectively, for 4 h to prepare FITC–HAPN–FBS complex. Cell culture medium was removed from the plate and replaced by 500 μL of FITC–HAPN–FBS complexes (0, 5, and 10%) complex together with 1.5 mL DMEM containing corresponding concentrations of FBS. After 4 h of treatment, MG-63 cells were washed with PBS and then fixed with 4% paraformaldehyde solution in PBS for 15 min at room temperature. Subsequently, cells were incubated with 1 ng/mL Hoechst 33258 for nuclei staining for 10 min. After washing several times with PBS, the internalization of FITC–HAPN–FBS complex was visualized by a laser scanning confocal microscope (LSM780, Carl Zeiss AG, Germany).

Cellular uptake detected by ICP-OES

The effect of FBS on HAPN internalization was determined by the phosphorus content of MG-63 cells after treated with 500 μL of HAPN–FBS (0, 2, 5, 8, and 10%) complexes in 1.5 mL of DMEM containing corresponding concentrations of FBS for 4 h. Cells were washed with PBS three times and then collected into PBS. Cell number of each sample was counted using a hemocytometer. Samples were subsequently treated with 20% nitric acid at 50 °C for 24 h to ensure that all nanoparticles were dissolved. The samples were diluted by adding 2 mL of DI water to ensure that the concentration of nitric acid is under 5%, and then free phosphorus content in each sample was analyzed by an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 4300 DV, Perkin Elmer, USA). The concentration of phosphorus standard was 0.01 mg/mL. Cells without nanoparticle treatment were used as the blank control.

Cytotoxicity of HAPN–FBS complex on cancer cells

MTT assay was carried out to measure the cell viability based upon converting MTT into formazan crystals only in living cells. MG-63 cells were seeded at a density of 5 × 103 cells/well into 96-well plate and cultured overnight. The medium was replaced by 200 μL of HAPN–FBS (0, 2, 5, 8, and 10%) complexes and pure DMEM with different concentrations of FBS (0, 2, 5, 8, and 10%) and cultured for 48 h. Then, 30 μL MTT was added to each well, and the 96-well plate was cultured in the incubator for another 4 h. The supernatant was removed in each well. Then, 200 μL DMSO was added to dissolve formazan crystals for another 10 min at 37 °C, and 150 μL of DMSO was subsequently transferred into a new 96-well plate to remove the particle precipitation. The optical density (OD) value of the new plate was measured by a microplate reader (Spark 10M, Tecan, Switzerland) at a wavelength of 492 nm.

Results and discussion

FBS protein adsorption on the surface of HAPNs

Morphological analysis of HAPN–FBS complex was carried out by TEM to directly reveal the FBS protein adsorption on HAPN surface. In the TEM images of HAPN–FBS (0%), the morphology of a single nanoparticle was clearly observed in rod shape with around 60 nm in length and 20 nm in width. Moreover, HAPNs without FBS exhibited clear and distinguishable surface in TEM images of HAPN–FBS (0%) complex (Fig. 1). By contrast, HAPNs became diffuse and seemed to be covered with a layer of gelatinous substance on the surface after combining with FBS. In TEM images of HAPN–FBS (5 and 10%) complex, especially in the image of HAPNs treated with 10%, the presence of a layer of substance was confirmed around the surface of HAPNs. The emerging layer derived from adsorption of FBS proteins was named protein corona. It is widely reported that protein coronas will form once nanoparticles enter into a biological system (Foroozandeh and Aziz 2015). For example, after being exposed to cell culture medium containing FBS, citrate-stabilized gold nanoparticles were coated by protein coronas and the density and thickness of coronas were highly dependent on the particle size (Piella et al. 2017).

Fig. 1
figure 1

TEM images of HAPN–FBS complexes with the FBS concentrations of 0, 5, and 10%

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ATR-FTIR spectroscopy was used here to confirm the protein adsorption on the surface of HAPN–FBS complex (Fig. 2a). The absorption bands of OH− ions (3645–3330 cm−1) and PO43− groups (563, 600, 632, 962, 1024, and 1088 cm−1) of the apatitic structure were observed in all the samples, including HAPNs and HAPN–FBS (0, 2, 5, 8, and 10%) complexes. The bands at 1455 cm−1 were attributed to a small amount of CO32− group formed during the open-air synthetic process. Noticeably, new bands appeared in the spectrum at 1651 and 1531 cm−1 corresponding to the amide I and amide II regions in HAPN–FBS (2, 5, 8, and 10%) complexes. Generally, the amide I band is located between 1700 and 1600 cm−1 and arises from C=O stretching vibrations of the peptide bonds. Amide II band is between 1600 and 1500 cm−1, originating from C-N stretching vibrations and N-H bending (Roach et al. 2005). This result suggested that HAPN surface was coated by proteins possibly derived from FBS.

Fig. 2
figure 2

(a) ATR-FTIR spectra of pristine HAPNs and HAPN–FBS complexes with varying FBS concentrations of 0, 2, 5, 8, and 10%. (b) QCM-D frequency changes in fifth harmonic upon rinsing with PBS (step A) and binding of only DMEM or DMEM with 10% FBS (step B) to HAPN surface

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In order to clearly verify this viewpoint, the real-time adsorption of cell culture medium and FBS on HAPN surface was investigated using QCM-D (Fig. 2b). HAPNs formed a layer on the QCM-D sensor using an electrophoretic deposition method. PBS solution firstly flowed past the surface of sensors with HAPNs to acquire an equilibrium state in step A. A significant decrease in frequency was shown in step B after HAPN layer was exposed to DMEM containing 10% FBS solution compared to DMEM alone. The frequency at 1500 s after adsorption of DMEM and DMEM with 10% FBS shifted to − 42.97 and − 78.44 Hz, separately. Compared with the adsorption of DMEM (152.11 ng increase of mass), the mass of HAPNs after adsorption of DMEM with 10% FBS increased 125.56 ng according to the Sauerbrey equation (Δm = −C × Δf/n, where C (mass sensitivity constant) = 17.7 ng cm−2 Hz−1 for 5 MHz crystals, n (overtone number) = 5). Thus, the adsorption of proteins on the surface of HAPNs upon contact with FBS was demonstrated combining the data of ATR-FITC.

Thermogravimetric analyses were carried out to further support the results of TEM, ATR-FTIR, and QCM-D that FBS proteins adsorbed on HAPN surface. Based on the result of TG profiles in Fig. S1, the initial mass loss of samples was around 4.3% derived from water desorption upon heating to 270 °C. Additional mass loss of around 10% was observed after the temperature rose from 270 to 530 °C in HAPN–FBS (10%) complex compared to HAPN–FBS (0%) complex, which was attributed to the decomposition of proteins from FBS. When the concentration of FBS increased from 2 to 10%, the mass loss of HAPN–FBS complexes showed a minor difference of around 3%. The protein adsorption was verified by TG as well.

Yang et al. indeed reported that nano-sized hydroxyapatite possessed strong affinity with proteins, such as bovine serum albumin (BSA)—the main component of fetal bovine serum (FBS) (Yang and Zhang 2009). Rod HAPNs exhibited relatively higher BSA adsorption in comparison with HAPNs in spherical and fibrous morphology (Swain and Sarkar 2013). It has been previously suggested that BSA can be adsorbed on the surface of hydroxyapatite through electrostatic attraction between the Ca2+ and PO43− of particles and COO− and NH3+ group of BSA (Wassell et al. 1995; Swain and Sarkar 2013). Consequently, these results in Figs.1 and 2 and Fig. S1 together confirmed the adsorption of proteins from FBS on HAPN surface possibly due to electrostatic force of attraction.

Improved dispersion and stability of HAPNs after FBS protein adsorption

In addition to the shape, size, and crystallinity of nanoparticles, it is generally accepted that protein corona is also a crucial factor in determining the property of nanoparticles in biological medium (Monopoli et al. 2011; Safi et al. 2011). The effect of FBS on HAPN dispersion was analyzed by DLS. After HAPNs were introduced into DMEM with 5% or 10% FBS and kept for 4 h without stirring, the hydrodynamic size of HAPNs was above 1700 nm (Fig. S2). The size of HAPNs in DMEM without FBS after stirring was above 1400 nm (Fig. 3b). It was demonstrated that FBS without stirring or stirring without FBS did not prevent the aggregation. Once HAPNs were added in DMEM containing different concentrations of FBS (0, 2, 5, 8, and 10%) and stirred for 4 h, the hydrodynamic size of HAPNs decreased greatly with the amount of FBS increased (Fig. 3a,b). Hence, stirring contributed to protein adsorption on HAPN surface and the formation of protein corona eventually resulting in the dispersion of HAPNs. The agglomerate size of HAPNs in DMEM without FBS was above 1400 nm, and the agglomerate size of HAPNs with 10% FBS was reduced to about 200 nm. However, 10% FBS could not completely prevent some HAPN agglomeration since the uncoated HAPN size observed from TEM was less than 100 nm. The most significant hydrodynamic size change of HAPNs happened when the concentration of FBS increased from 0 to 2%.

Fig. 3.
figure 3

Hydrodynamic size distribution (a) and average hydrodynamic size (b) of HAPN–FBS complexes at different concentrations of FBS. HAPN concentration was kept at 1000 μg/mL, and the HAPN suspensions were stirred in DMEM with FBS (0, 2, 5, 8, and 10%) for 4 h before DLS particle size measurements. Error bars indicate s.d. (n = 3)

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The agglomeration state of HAPNs influenced by FBS was examined by inverted microscope and SEM to support the DLS data. In the microscopy image in Fig. 4 (top row), uncoated HAPNs in DMEM (0% FBS) appeared as large clumpy structures. With increasing concentrations of FBS, particle dispersion improved and the size of the agglomerates significantly decreased up to 5% FBS, after which the dispersion effect did not appear to increase further.

Fig. 4
figure 4

Top row: microscopy images (100×) of HAPN–FBS complexes with varying FBS concentrations of 0, 2, 5, 8, and 10% after standing for 2 h in a 96-well plate. Bottom row: SEM images of HAPN–FBS complexes with varying FBS concentrations of 0, 2, 5, 8, and 10% after filtration (black holes refer to the pores of membrane filters)

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Membranes with 1 μm pores (black holes in SEM images in Fig. 4) were applied to filter the HAPN–FBS complex for SEM observation. The resultant agglomerate size of uncoated HAPNs (0% FBS) was above 1 μm (Fig. 4, bottom row). The presence of FBS turned those micrometer-sized particles into smaller clumps. In SEM images, it was apparent that most of the compacted and large particles disappeared after the addition of FBS. When the concentration of FBS increased to 5%, the particles were in a relatively loose and flaky state. However, there still existed some thin HAPN aggregates demonstrating that the FBS protein adsorption could not eliminate the aggregation thoroughly.

The stability of HAPNs in DMEM with different concentrations of FBS was measured by DLS. The agglomerate size of uncoated HAPNs (0% FBS) and HAPN–FBS (2% FBS) increased substantially within 90 min from 1600 and 800 nm to 4300 and 3400 nm. In contrast, the agglomerate size of HAPNs mixed with 8 and 10% FBS essentially remained unchanged and less than 300 nm over 90 min (Fig. 5a). The agglomerate size of HAPNs was reduced to less than 200 nm in DMEM with 10% FBS and remained at 200 nm over 5 days (Fig. 5b). Therefore, FBS not only decreased the agglomerate size of HAPNs but also maintained the stability of HAPN for up to 5 days.

Fig. 5
figure 5

(a) Average hydrodynamic size of HAPN–FBS (0, 2, 5, 8, and 10%) complexes in a short time and (b) HAPN–FBS (10%) complex after storage at 37 °C for 0, 1, 2, and 5 days. HAPN concentration was kept at 1000 μg/mL. The average hydrodynamic size in panels (a) and (b) was measured by DLS. Error bars indicate s.d. (n = 3)

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The impact of serum on hydrodynamic particle size has been reported in other inorganic nanoparticles. Serum could disperse the agglomerates of cerium oxide and zirconium oxide nanoparticles into ultrafine fractions (Schulze et al. 2008), and a concentration of only 1% of FBS was found to be sufficient for dispersing titanium dioxide nanoparticles (Ji et al. 2010). In our study, the agglomeration state of HAPNs changed sharply after addition of 2% of FBS. The low concentration of FBS apparently played a prominent role in dispersing HAPNs, although the most highly dispersed HAPN suspension was observed with 10% FBS. The FBS in physiological environment is a key factor in determining the dispersion of these nanoparticles. In the battle between van der Waals attractive forces with electrostatic repulsive forces and steric repulsion, normally, agglomeration occurs if the attractive van der Waals forces prevail against the repulsion of the nanoparticles (Thomas et al. 2015). Hydroxyapatite nanoparticles were found to agglomerate in cell culture medium without serum (Bauer et al. 2008). Here, the zeta potential of HAPNs was − 7 mV, while the zeta potential of HAPN–FBS (10%) complex was − 23 mV. Adsorption of proteins from FBS on the HAPN surface likely provided sufficient steric and electrostatic repulsion to overcome van der Waals attraction forces between the particles.

Cellular uptake of HAPN–FBS complex in MG-63 cells

Nanoparticles will selectively adsorb biomolecules like proteins and rapidly form a corona in biological fluids, which provides the biological identity of the inorganic materials (Monopoli et al. 2012). In addition to the influence of protein corona on nanoparticle agglomeration, it is now commonly believed that the protein corona will greatly influence the interaction of nanoparticles with cells and the fate of the nanoparticles inside the cells (Hadjidemetriou and Kostarelos 2017). To investigate cellular uptake affected by FBS, FITC-HAPNs were prepared to visualize the nanoparticles in the cells under a laser scanning confocal microscope. It was proved that the average hydrodynamic sizes of FITC-HAPNs after treatmented with 0, 5, and 10% FBS were 1670, 483, and 402 nm, respectively, in Fig S3. Although the reduction of the size of FITC-HAPNs was not as significant as HAPNs after the addition of FBS, the dispersion state of FITC-HAPNs still improved with increasing FBS concentration. The confocal images in Fig. 6a demonstrated that the FITC-HAPNs were internalized by MG-63 cells. The amount of FITC-HAPNs internalized in MG-63 cells after 4 h incubation increased with elevated FBS levels as indicated by more intense green fluorescence signals.

Fig. 6
figure 6

Cellular uptake of FITC–HAPN–FBS complex (a) and HAPN–FBS complex (b) at different concentrations of FBS in MG-63 cells after 4 h treatment. Cells were treated with FITC–HAPN–FBS complex or HAPN–FBS complex at the HAPN concentration of 500 μg/mL, and HAPN internalization was evaluated using confocal laser scanning microscope (a) or ICP-OES (b) to determine intracellular phosphorus content. Green signals represented FITC–HAPN–FBS complex and blue signals represented cell nuclei. * equals p < 0.05 when compared to the phosphorus content of MG-63 cells treated with HAPNs in DMEM at the FBS concentration of 0%. Error bars indicate s.d. (n = 3)

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The cellular uptake of HAPN–FBS was also measured by ICP-OES based on the phosphorus concentration in MG-63 cells (Fig. 6b). The alteration of phosphorus content in cells after being exposed to HAPN–FBS (0, 2, 5, 8, and 10%) complexes was consistent with the FITC fluorescence results. The internalized HAPNs increased with FBS coating levels, with the amount of cellular uptake of HAPN–FBS (10%) complex 1.47-fold greater than uncoated HAPNs (0% FBS). However, the intracellular HAPNs amount exhibited significant increase only after FBS concentration reached 8%, which represented that FBS did not show a big impact on HAPN internalization compared to particle dispersion.

The results from laser scanning confocal microscope and ICP-OES both revealed the enhanced uptake efficiency of HAPNs with the addition of FBS. Such a higher cell internalization might be caused by the formation of the protein corona, or due to a decrease of the agglomeration state, or even the combination of both effects. Some other studies also reported the role of pre-coating nanoparticles with proteins in targeting efficiency and nanoparticle uptake. The uptake amount of functionalized gold nanoparticles modified by different aromatic thiols was similar after they were coated with serum, demonstrating the serum coating played a major role in internalization regardless of the surface functional groups (Park et al. 2011). In addition, the level of cellular uptake was found strongly dependent on the agglomeration state for silica nanoparticles (Halamoda-Kenzaoui et al. 2017).

Cytotoxicity of HAPN–FBS complex

Cell viability of MG-63 cells after 48 h treatment of HAPN–FBS complex was examined by MTT assay to investigate the influence of FBS on HAPN cytotoxicity (Fig. 7). The effect of serum starvation was also evaluated to uncover the real cytotoxicity of HAPN–FBS complex. After incubation with low concentrations of FBS (0 and 2%), the viability of MG-63 cells decreases to 62 and 89%, respectively. When the concentration of FBS was above 5%, no obvious inhibition on the proliferation of MG-63 cells was found. HAPN–FBS (0, 2, 5, 8, and 10%) complexes resulted in comparable inhibition rate of about 60% on cell proliferation. Considering the toxicity due to the lack of FBS, the cytotoxicity of HAPN–FBS (0 and 2%) complexes was actually lower than that of HAPN–FBS (5, 8, and 10%) complexes after 48 h exposure. The cytotoxicity of HAPN–FBS (5, 8, and 10%) complexes actually did not show an obvious difference. In conclusion, the hydroxyapatite nanoparticles with protein coronas can inhibit the proliferation of osteosarcoma cells more efficiently than the pristine nanoparticles.

Fig. 7
figure 7

Viability of MG-63 cells after being exposed to DMEM with varying FBS concentrations of 0, 2, 5, 8, and 10% with or without HAPN–FBS complex at the HAPN concentration of 1000 μg/mL for 48 h. Cell viability was normalized to that of MG-63 cells cultured in DMEM with 10% FBS but without HAPNs. * equals p < 0.05 when compared to the cell viability of MG-63 cells treated with 10% of FBS. Error bars indicate s.d. (n = 3)

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It was reported that pre-incubation with protein formed the corona around silver nanoparticles and ultimately changed overall toxicity (Durán et al. 2015). In addition, a study on modified silica nanoparticles drew the conclusion that both the protein corona and the agglomeration kinetics should be taken into account in terms of cytotoxicity of nanoparticles (Mortensen et al. 2013). Such discovery highlights the importance that FBS in culture medium may modulate nanoparticle toxicity. The slightly increased nanotoxicity of HAPNs after FBS protein adsorption might depend not only directly on the formation of protein coronas but also on the improvement of particle dispersion caused by protein adsorption.

In summary, the FBS protein adsorption and formation of protein coronas on HAPN surface were confirmed. Figure 8 illustrates how the addition of FBS in cell culture medium improved the dispersion of HAPNs in an FBS concentration-dependent manner. Nevertheless, unlike the agglomeration of HAPNs, the cellular uptake and cytotoxicity of HAPNs increased slightly with FBS supplement but did not undergo a significant change.

Fig. 8
figure 8

Illustration of how the adsorption of FBS proteins on the HAPN surface influenced the agglomeration state of HAPNs and subsequent cell–nanoparticle interaction

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Conclusion

In this study, we obtained clear evidence of the adsorption of FBS proteins on hydroxyapatite nanoparticle surface to form protein coronas. The presence of FBS in cell culture medium decreased the agglomeration state of HAPNs. An FBS concentration as low as 2% was sufficient to achieve well-dispersed HAPN suspension. The agglomerate size of HAPN–FBS (10%) complex was significantly reduced to 200 nm and was maintained at that size for at least 5 days. In contrast, HAPNs in the medium without FBS were highly unstable and developed massive agglomerates (1400 nm). In particular, the adsorption of FBS on nanoparticles led to a slight increase in cellular uptake and tumor cell cytotoxicity but not as significantly as enhanced particle dispersion. Overall, these findings emphasize the potentially enormous impact of FBS in biological environment on the performance of HAPNs.