Preparation and functional study of pH-sensitive amorphous calcium phosphate nan

Baolong Niu,Min Li,Jianhong Jia,Lixuan Ren,Xin Gang,Bin Nie,Yanying Fan,Xiaojie Lian,Wenfeng Li,*

1 College of Materials Science and Engineering,Taiyuan University of Technology,Taiyuan 030024,China

2 Key Laboratory of Interface Science and Engineering in Advanced Materials,Taiyuan University of Technology,Taiyuan 030024,China

3 Department of Biomedical Engineering,Research Center for Nano-biomaterials &Regenerative Medicine,College of Biomedical Engineering,Taiyuan University of Technology,Taiyuan 030024,China

4 Shanxi Key Laboratory of Material Strength &Structural Impact,Institute of Biomedical Engineering,Taiyuan University of Technology,Taiyuan 030024,China

5 School of Basic Medical Sciences,Shanxi Medical University,Taiyuan 030001,China

Keywords:Hyaluronic acid Amorphous calcium phosphate Fluorescent carbon nanoparticles pH-sensitive Tumor targeting

ABSTRACT Recently,multifunctional nanoparticles have shown great prospects in cancer treatment,which have the ability to simultaneously deliver the drug,image and target tumor cells.In this paper,we designed a luminescent nanoparticles platform based on hydrothermal hyaluronic acid/amorphous calcium phosphate(HA-FCNs/ACP)with multifunctional properties for drug delivery,bio-imaging,and targeting treatment.HA-FCNs/ACP shows an ability to load curcumin (Cur) with pH-sensitive responsive drug release behavior and excellent biocompatibility.HA-FCNs/ACP dispersed in the cytoplasm through the overexpressed CD44 receptor that is actively targeted into human lung cancer cells (A549 cells).Meanwhile,the viability of A549 cells was significantly inhibited in vitro.The prepared HA-FCNs and HA-FCNs/ACP both exhibit excellent targeted bioimaging performance on cancer cells.Hence,the as-prepared nanoparticles have promising applications in treating tumor disease.

1.Introduction

Target bio-imaging and delivery are promising new approaches for the study and treatment of a variety of diseases,including,most notably,cancer[1,2].Therefore,exploring an intelligent multifunctional nanoplatform to realize tumor targeting,on-demand sensitivity therapy,and real-time bio-imaging is a promising prospect for the successful treatment of tumors.Over the past several years,fluorescent nanocarrier based on metal,silica,polymers,semiconductors,etc.have great promising in the field of biosensors and bioimaging probes due to their excellent biostability as well as sensitivity.Inspired by the electronic response of carbon dots(C-dots) and the toxicity of metal-based quantum dots (QDs) [3],fluorescent carbon nanoparticles have been intensively studied for the insightful applications in fluorescent probes,lightemitting devices,and biosensors,which is due to their excellent factors on the basis of low-toxicity,environmentally-friendly alternatives and biocompatibility.Jeonget al.[4]employed mango fruit to prepare water-soluble and non-toxic bio-imaging fluorescent carbon nanoparticles (FCNs),derived by controlled carbonization method.In addition,the appropriate functionalization of FCNs is benefit for deriving novel composite materials for various unique applications.

Hyaluronic acid (HA) is a nontoxic and biodegradable natural acidic polysaccharide macromolecule with unique viscoelasticity and limited immunogenicity,which plays important roles in the organization of the extracellular matrix (ECM),as well as cell motility,adhesion and differentiation.It is also found abundantly overexpressed CD44 receptor that binds to the HA ligand in uncontrolled growing non-differentiated tumor angiogenesis,thus it could be used as a targeting tumor matrix [5].Moreover,hyaluronidase (HAase) can hydrolyze HA into low molecular weight fragments that is an endoglycosidase in the cell,of which approximately 1 × 105to 2 × 105IU is taken up through endocytosis in many cancer cells or 3-7-fold in normal cell,which is important for cancer diagnosis and therapy based on its specific response to the tumor environment [6].Due to these properties,it has been extensively studied in biomedical fields,ranging from tissue engineering to drug delivery [7-9].Compared to pure form,HA modified organic or inorganic hybrid materials have attracted more attention of scholars[10,11].Up to now,amorphous calcium phosphate(ACP),a metastable phase[12],has attracted increasing attention in biomedical fields due to the absence of both foreign body response[13]and toxic byproducts[14],large specific surface area,high drug loading capacity,as well as controlled drug release behaviour [15,16],which is compared with other calcium phosphate materials.In addition,the solubility of ACP nanomaterials increases with the decrease of pH value in aqueous solution,according to which can be used as pH-sensitive drug carriers for simultaneously responding to the tumor micro-environment to realize on-demand stimuli-responsive drug release [17,18],thus avoiding secondary effects or general toxicity.In the present work,we have been committed to developing a drug delivery system conjugating fluorescent carbon particles (HA-FCNs) offering bioimaging and active targeting properties with ACP for multiple applications such as in drug delivery,metal ion sensing and tissue engineering.

Curcumin(Cur)is an acidic polyphenol obtained from turmeric,with a variety of pharmacological effects such as anticancer,anti-HIV and antibacterial.In recent years,curcumin has become a promising antitumor drug due to its relatively few side effects and high safety.Its mechanism of action mainly includes inhibition of tumor invasion and migration,induction of tumor cell apoptosis and inhibition of various cell signaling pathways.Despite the good antitumor effects of curcumin,its low bioavailability,poor solubility in aqueous solution and rapid degradation under physiological conditions make clinical administration difficult.It is necessary to design a drug delivery system based on the structural characteristics of curcumin,for example,Cur was physically encapsulated within carrier to improve its solubility and targeted to the tumor site for release,thus improving its bioavailability at the lesion site and giving full play to its role in tumor therapy.

Herein,we obtained fluorescent carbon nanoparticles (HAFCNs) fabricated by hydrothermal,synthesized HA-FCNs/ACP nanospheres using the coprecipitation method and prepared drug-loaded nanoparticles (Cur@HA-FCNs/ACP) were by coblending and co-precipitation methods.In addition,the encapsulation and loading efficiency of Cur,pH sensitivity,tumor targeting and cooperative image-guided and the other properties of HAFCNs/ACP nanoparticles were evaluated.In particularly,to investigate the targeting performance of the carrier,we used A549 cells enriched with high expression of CD44 receptor on the surface and MG63 cells that do not express it.Hyaluronic acid can actively target to CD44 receptor on the surface of cancer cells,enter cancer cells through endocytosis,and release the antitumor drug Cur under the stimulation of pH acidic environment,thus achieving targeted drug release.As a control,the carrier could not target to normal cells MG63 cells,further illustrating the tumor-targeting performance of the carrier.

2.Materials and Methods

2.1.Materials

Hyaluronic acid [HA,(C14H20NNaO11)n,80-2000 kDa,BR] and phosphate buffer saline (PBS,0.01 mol·L-1,pH 7.2-7.4) were supplied from Beijing Solarbio Science &Technology Co.,Ltd.Curcumin (99%) was obtained from Shanghai Aladdin Biochemical Technology Co.,Ltd.Calcium chloride anhydrous(CaCl2),diammonium hydrogen phosphate [(NH4)2HPO4],sodium hydroxide(NaOH) and dialysis membrane (molecular weight cutoff of 3500)were purchased from Sinopharm Chemical Reagent Co.Ltd.All the other reagents obtained from Tianjin Kemiou Chemical Reagent Co.,Ltd.were analytical grade and all solutions were prepared by deionized water.

Human lung cancer cell line (A549) and human osteosarcoma cell line (MG63) were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences.Fetal Bovine Serum(FBS)and trypsin were available from TransGen Biotech(Beijing)Co.,Ltd.RPMI Medium 1640 and MEM Alpha were purchased from Gibco BRL (Carlsbad,CA,USA).Live &Dead viability/cytotoxicity assay kit for animal cells was obtained from Jiangsu KeyGen Biotech Co.,Ltd.

2.2.Synthesis and characterization of fluorescent carbon nanoparticles

The process of HA-FCNs is schematically shown in Fig.1 (a).0.2 g of hyaluronic acid was dissolved in deionized water and then was placed in Teflon liner before reacted in muffle furnace.After reacting for 3 h,the solution was subjected to dialysis (molecular weight cutoff of 3500)and was followed by freeze-drying in order to obtain HA-FCNs.

2.3.Synthesis of fluorescent carbon nanoparticles decorated amorphous calcium phosphate

HA-FCNs/ACP nanoparticles were formed by coprecipitation method [19],as shown in Fig.1 (b).For the synthesis of nanoparticles,calcium chloride anhydrous (CaCl2) and diammonium hydrogen phosphate [(NH4)2HPO4] were taken as precursors of Ca2+and PO3-4 ions.Briefly,HA-CDs were dissolved in 30 ml of deionized water.In the meanwhile,aqueous solutions of CaCl2(0.073 mol·L-1) and (NH4)2HPO4(0.024 mol·L-1) were also prepared in different beakers.Then,CaCl2was added dropwise to the solution of HA-FCNs,stirred at 30°C for 1 h.After that,(NH4)2-HPO4was added slowly into the above solution and made alkaline(pH 8)by adding NaOH with continuously stirring for 10 min.Ultimately,the final product was collected by centrifugation and followed by cryodesiccation.

2.4.Characterization

The crystalline phase of HA,HA-FCNs,ACP and HA-FCNs/ACP nanomaterials were characterized by X-ray diffraction (XRD,Rigaku D/max 2500 V) with Cu-Kα radiation (λ=0.154178 nm).The functional groups of samples were recorded by FTIR (Model:Nicolet-710 spectrometer) at wavelengths ranging from 500 to 4000 cm-1using KBr pellet.The microstructure,size and element distributions of samples were examined by Scanning electron microscope (SEM,FEI Magellan 400,USA) and transmission electron microscope(TEM,JEM2100F,Japan),respectively.The fluorescent images were performed on an inverted biologic microscope(Motic AE31).The UV-vis spectroscopy was carried out on a UVvis spectrophotometer (UV1800,Shimadzu Corporation) in the wavelength range of 200-500 nm.The size and zeta potential of samples were measured using dynamic light scattering (DLS,Malvern,UK).The elemental compositions were obtained by XPS(Thermo Scientific ESCALAB 250Xi,USA).Photoluminescence (PL)spectra were determined from a Fluoro Max-4 fluorometer(Horiba Jobin Yvon Inc,France).The confocal images of cells were taken on a confocal laser scanning microscope (CLSM,C2 Plus,Nikon).

2.5.Curcumin (Cur) loading and release study

For drug loading,the Cur-loaded nanoparticles were prepared by blending method and coprecipitation method [20].Cur(5 mg·ml-1) was mixed with the solution of HA-FCNs and CaCl2while the suspension was stirred at 30 °C.After 1 h,(NH4)2HPO4was added into this solution gently stirred for another 10 min and then centrifuged to collect free Cur and Cur@HA-FCNs/ACP.The drug encapsulation efficiency (EE) and loading capacity (LC)were obtained using the following equation:

Drug release was studied in phosphate buffered saline(PBS,pH 7.4) and acetate buffer saline (ABS,pH 5.4),which were as drug release mediums to simulate normal blood/tissue and tumor surroundings,using a membrane dialysis method at 37 °C.The Curloaded HA-FCNs/ACP nanoparticles dispersed in release mediums were placed in pretreated dialysis bags (molecular weight cut off of 14000),which were immersed in beakers at 37°C under shaking.At predetermined time intervals,the medium was withdrawn for measuring at 424 nm by UV-vis and replenished with the same volume of fresh medium.And the whole process needed to be shielded from light.

2.6.Cytotoxicity assay and Live &Dead viability assay

The cytotoxicity assays of HA-FCNs and HA-FCNs/ACP were measured on A549 and MG63 cells.In brief,200 μl of cells,at a density of 2.5 × 104cells·ml-1,were plated on a 96-well plate and then incubated for 24 h at 37°C in a humidified 5%CO2atmosphere.To determine cell viability,the cells were treated with different concentrations of HA-FCNs or HA-FCNs/ACP dissolved in the medium for another 24 h.The medium containing HA-FCNs or HAFCNs/ACP was replaced with 90 μl of fresh medium and 10 μl of MTT,after which the cells were incubated for 4 h.Finally,the medium was removed and 200 μl of DMSO was added to the wells,shaking gently for 10 min.The absorbance was measured at 492 nm using an Enzyme marker analyzer(SM-3,China)and compared with the result of the control well.Regarding Live &Dead viability assay,the medium was replaced with 200 μl of pretreated fluid containing Calcein AM and Propidium Iodide (PI) for 40 min after the cells were treated 24 h.The cell images were followed by measurements using a fluorescence microscope.The magnification was 10 times the original size.

Fig.1.Schematic presentation of synthetic procedure of HA-FCNs(a)and HA-FCNs/ACP (b).

2.7.In vitro cell imaging

HA-FCNs and HA-FCNs/ACP incubated cells were analyzed by confocal imaging.A549 and MG63 cells were seeded into a 24-well plate,at a density of 2.5× 104cells·ml-1,and then incubated at 37°C in a humidified 5%CO2atmosphere for 24 h.To observe the effect of cell imagingin vitro,the cells were treated with HA-FCNs and HA-FCNs/ACP at concentrations of 0.01 mg·ml-1and 0.05 mg·ml-1for 2 h,respectively.After that,the cells were washed several times with PBS and then fixed with paraformaldehyde for a predetermined period time.Then,paraformaldehyde was discarded and washed with PBS three times.Finally,fluorescence images were captured using a confocal laser scanning microscope.

3.Results and Discussion

3.1.Characterization of HA-FCNs

3.1.1.Structural characterization of HA-FCNs

Fig.2(a) showed that the fluorescence intensity of HA-FCNs shows a trend of enhancement and then decrease with the increase of excitation wavelength,which may be caused by the different luminescent sites on the surface of carbon quantum dots.The characteristic spectra of prepared HA-FCNs were shown in Fig.2(b) to understand the capability of fluorescence emission,which can be seen that the absorption peak at 256 nm is caused by the absorption of π-π* transition formed by CC group.In addition,the maximum emission peak is located at 460 nm for the emission spectrum with an excitation wavelength of 380 nm.Under excitation with a lamp at 365 nm,HA-FCNs strongly emitted a blue color(Fig.2(c)).XPS was then performed to evaluate the elemental compositions of HA and HA-FCNs,shown in Fig.3(a).The full XPS spectra of HA and HA-FCNs exhibit seven peaks at 285.0,400.0,507.0 and 533.0 eV,which correspond to C1s,N1s,Na1s and O1s,respectively [21].The presence of Na1s is due to the certain sodium element in HA.As shown in Table 1,the increase in the relative content of carbon elements in HA-FCNs confirm that HA was partially carbonized at some level.Moreover,the high-resolution C1s scans of HA-FCNs,presented in Fig.3(b),demonstrate three different chemical bonds of C=C,C-C,and O-C=O around 284.25,285.33 and 284.34 eV.To determine their size and shape,TEM images,as shown in Fig.3(c) and (d),exhibit that the small spherical particles of as-prepared HA-FCNs have a size of less than 10 nm,and the latticed-spacing of HA-FCNs is 0.33 nm indicated from Fig.3(d),which is graphene-like structures.

Table 1 The relative elemental compositions of HA and HA-FCNs

3.1.2.Cytotoxicity study and cell imaging

The cytotoxicity of HA-FCNs with A549 and MG63 cells were subsequently monitored using cell viability assay.The results of MTT assay are shown in Fig.4(a),which were carried out against A549 and MG63 cells.To check the cytotoxicity of HA-FCNs in the concentration range from 0 to 1.2 mg·ml-1,A549 and MG63 cells were incubated for 24 h.According to Fig.4(a),the cell viability of both A549 and MG63 cells was higher than 80%after incubation with HA-FCNs for 24 h,respectively.In addition,the results of HA-FCNs for MG63 cells basically showed a concentrationdependent increase in cytotoxicity,however even when the HAFCNs concentration was increased to 1000 μg·ml-1,the cell viability rate was still greater than 80% and the nanoparticles did not show significant cytotoxicity,indicating that HA-FCNs with great biocompatibility.Further,the cell survival rate of A549 cells appeared higher than 100% after incubation with HA-FCNs for 24 h under certain concentrations,which was mainly due to the HA in HA-FCNs can be specifically identified by CD44 receptors on the surface of tumor cells and thus enter the inside of tumor cells through endocytosis.In conclusion,HA-FCNs showed low cytotoxicity and were fairly safe to be used as drug carriers.

Fig.2.(a)PL spectra of HA-FCNs at different excitations(280-400 nm).(b)Characteristic optical spectra of HA-FCNs produced.(c)Digital images of HA and HA-FCNs under 365 nm UV lamp.

Fig.3.X-ray photoelectron spectroscopy (XPS) spectra of survey spectra (a) of HA and HA-FCNs.The C1s peaks of HA-FCNs (b).Transmission electron microscope (TEM)images of HA-FCNs,showing size and shape (c) and (d).The scale bars are 10 nm and 2 nm,respectively.

To further evaluate the cytotoxic effects of HA-FCNs,Live &Dead cell viability assays were utilized [22].Effect of HA-FCNs on A549 and MG63 cells morphology was noticed from images seen under an inverted biologic microscope,presented in Fig.4(b).As shown in Fig.4(b),the cell morphology of both A549 and MG63 cells was basically unchanged after 24 h incubation with HAFCNs,and the cells remained in a good physiological state,which was consistent with the results of MTT experiments,the cell viability is still ＞80%,clearly indicating that HA-FCNs is an ideal material with the advantage of good biocompatibility.

Fig.4.(a)Cytotoxicity tests of the as-prepared HA-FCNs prepared using A549 and MG63 cells.(b)Live&Dead cell viability assays of HA-FCNs on A549 and MG63 cells,kept the magnification at 10×.And the concentrations of HA-FCNs were 0,100 and 1000 μg·ml-1 from left to right,respectively.

Fig.5.Confocal microscope images of A549 and MG63 cells treated with HA-FCNs at 0.01 mg·ml-1.The scale bars both are 50 μm.

Visualization of the fluorescence level in cells allows the evaluation of the cellular interactions for advance biomedical applications [23].To evaluate the cellular uptake of HA-FCNs andin vitrocell imaging performance,A549 and MG63 cells were treated with samples,as well as monitored with a confocal laser scanning microscope.As shown in Fig.5,stronger fluorescence signals were detected from A549 cells than in MG63 cells,when they were examined at their corresponding excitation wavelengths.Meanwhile,the presented fluorescence color was consistent with that of Fig.2(c),while no obvious fluorescence signal was observed in MG63 cells,indicating that HA-FCNs could not enter MG63 cells by active targeting,suspended in solution and was removed by PBS washing before observation.In contrast,a stronger fluorescence signal was observed in A549 cells,which was attributed to the fact that HA-ACP could actively target the CD44 receptor overexpressed on the surface of A549 cells and thus enter the cancer cellsviaendocytosis.These results suggested that HA-FCN can actively target to cancer cells,and thus realize the targeted bioimaging function of carbon quantum dots on cancer cells.

3.2.Characterization of HA-FCNs/ACP nanoparticles

There is no doubt that ACP is a metastable phase of hydroxyapatite(HAP),and it has been demonstrated that amorphous calcium phosphate has better biodegradability than hydroxyapatitein vivo[24].The combination of ACP with HA-FCNs to carry Cur represents an exciting advancement in cancer treatment.

3.2.1.Preparation of HA-FCNs/ACP nanoparticles

As expected,the nanoparticles emitted a blue color after illumination with a lamp at 365 nm,presented in Fig.6(a),which is consistent with HA-FCNs (Fig.2(c)).The crystal phases of ACP before and after decorated by HA-FCNs are characterized using XRD.As shown in Fig.6(b),the XRD of ACP shows a broad diffraction line at 30°that is exhibited a typical amorphous phase of calcium phosphate[19],while the results of the XRD of the HA-FCNs/ACP is consistent with the XRD of ACP.

The coprecipitation formations of ACP and HA-FCNs/ACP were further depicted from FTIR for chemical composition,as shown in Fig.6(c).In the spectra of ACP and HA-FCNs/ACP,the band at around 1620 cm-1assigned the C=O stretch for carboxylic acids of HA-FCNs is shifted to a higher wavenumber at 1640 cm-1indicating the electrostatic interaction between Ca2+and carboxyl groups of HA-FCNs[25].Due to carboxyl groups,HA-FCNs showed a highly negative charge irrespective of the pH(presented in Fig.6(d)),providing further evidence to support this analysis.While the unresolved absorption bands at around 572 and 1047 cm-1are assigned to the characteristics ofMoreover,the band at around 3430 cm-1,attributed to the adsorbed water of ACP,and the adsorption peak located at 3400 cm-1ascribed to the Hbonded hydroxyl group of HA-FCNs are overlapped.The small peak at 875 cm-1proves the presence ofin ACP and HA-FCNs/ACP nanoparticles.In addition,both the samples before and after loading HA-FCNs have the ν2 vibrational mode of carbonate ion at 873 cm-1,as well as the ν3 vibrational mode of carbonate ion at 1460 and 1419 cm-1,indicating that the presence ofgroup in ACP and HA-FCNs/ACP nanoparticles,which may be derived from the dissolved CO2from atmosphere [26-28].

Fig.6.Digital images of ACP and HA-FCNs/ACP nanoparticles under 365 nm UV lamp(a).The X-ray diffraction(XRD)patterns in the 2θ range 10°-80°of ACP and HA-FCNs/ACP (b).FTIR spectra infrared spectra of HA-FCNs,ACP and HA-FCNs/ACP nanoparticles (c).Zeta potential of HA-FCNs as a function of pH in an aqueous dispersion (d).

Fig.7.SEM image of HA-FCNs/ACP nanoparticles synthesized by coprecipitation method,which the magnifications is ×30000 (a).Elemental map distributions (b)-(f) for some of the elements contained in the synthetic standard HA-FCNs/ACP nanoparticles shown by scale bars of 100 nm.Particle size distribution of HA-FCNs/ACP nanoparticles by DLS (g).

The microstructure and size of the as-prepared HA-FCNs/ACP nanoparticles were investigated with SEM,EDS and DLS,presented in Fig.7.In the HA-FCNs/ACP nanocomposite(inset of Fig.7(a)),the spherical nanoparticles show uniform sizes,observed from SEM,which range from 80 to 120 nm.Fig.7(b)-(f)shows the distribution maps for some elements present in HA-FCNs/ACP nanocomposites.As it can be appreciated,Ca,P,C,N and O are quite homogeneously distributed,indicating that HA-FCNs are evenly dispersed in ACP.But,the mean diameter of HA-FCNs/ACP nanomaterials measured using DLS is about 172 nm,shown in Fig.7(g),which is different from the SEM results(80-120 nm).The possible reason for this difference is the variation in the sample preparation method,where in the DLS test the sample is in an aqueous solution and the particle size distribution is directly observed,while in the SEM test the specimen is in a dry state.

3.2.2.Assessing release of drug

To investigate the release kinetics of Cur from Cur@ HA-FCNs/ACP,we first studied release behaviors of Cur in PBS buffers under different pH values(pH=5.4 and 7.4)with constant shaking,which is shown in Fig.8(a).It can be seen that the different release of Cur from the Cur@HA-FCNs/ACP nanoparticles,collected at different time intervals to evaluate the pH responsive drug release.In the natural environment (pH 7.4),61% Cur was released from Cur@HA-FCNs/ACP after 20 h.Nevertheless,it was noted that the cumulative dissolution of Cur in the early stage (2 h) reached 34.4%,with no obvious release.At last,the dissolution test finished after the continuous release of Cur in 25 h.However,the release rate was faster in the acidic buffer(pH 5.4)than in the pH 7.4 environment,and the cumulative release rate of Cur reached 81%,which was greater than that in the pH 7.4 buffer (～61%).This may due to the gradual degradation of the carrier under acidic conditions,which contributing to the slow release of the loaded drug.This revealed that the synthesized carrier is pH-sensitive.

The results above indicated that the Cur@HA-FCNs/ACP are likely to be a highly promising drug delivery system that can control the release of anticancer drug smartly and improve the therapeutic efficacy.

3.2.3.Cell viability and cell imaging

In order to test this anticancer ability,the drug loading capability of HA-FCNs/ACP to deliver Cur was investigated against A549 cells.Here,the possible cytotoxicity of samples was evaluated by measuring cell viability using an MTT assay.As expected,the cytotoxicity increased with increasing HA-FCNs/ACP before and after loading Cur concentration.As shown in Fig.8(b),HA-FCNs/ACP nanoparticles have good biocompatibility and retain minimal effects on cell viability and proliferation,according to more than 90% of cell viability at a high concentration (32 μg·ml-1).By comparison,the cell inhibition rate of Cur@HA-FCNs/ACP was greatly highly than that of HA-FCNs/ACP unloading Cur and significantly influenced in time-and concentration-dependent manner,suggesting that cell activity is apparently inhibited by Cur released from Cur@HA-FCNs/ACP.The results also reflected the potential of HA-FCNs/ACP for A549 cells growth inhibition.

The CLSM images showed characteristic blue emission for both A549 and MG63 cells,presented in Fig.9.Depending on the type of cells,the imaging results of HA-FCNs/ACP are corresponding to that of HA-FCNs,the HA-FCNs/ACP had stronger fluorescence signals on the A549 cells compared to MG63 cells,which revealed the targeted bioimaging function of HA-FCNs/ACP on the tumor cells,indicating that ACP decorated by HA-FCNs has detection and targeting abilities,appearing to revolutionized cancer diagnosis and therapy.Further advances in the understanding of HAFCNs/ACP will provide new opportunities,especially for cancer treatment.

4.Conclusions

In this report,fluorescent carbon nanoparticles(HA-FCNs)were successfully synthesizedin situ,exhibiting excellent targeted bioimaging effects.In addition,fluorescent carbon nanoparticles/amorphous calcium phosphate (HA-FCNs/ACP) nanoformulations were fabricatedviaco-precipitation method,in which CaCl2as the calcium source and (NH4)2HPO4was used as the phosphorus source.The experiments showed that the as-prepared HA-FCNs/ACP nanoparticles had excellent biocompatibility,targeted bioimaging effects,and favorable pH-responsive drug release property.Hence,the synthesized HA-FCNs/ACP nanocomposite might provide a great potential for integrated diagnostic and therapeutic applications.

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.

Acknowledgements

The work was financially supported by the National Natural Science Foundation of China (31700689),Natural Science Foundation of Shanxi Province (201901D111115),Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (172040098-S),and Transformation of Scientific and Technological Achievements Programs of Higher Education Institutions in Shanxi (2020CG015).