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Biodegradable Drug-loaded Hydroxyapatite Nanotherapeutic Agent for Targeted Drug Release in Tumors Wen Sun, Jiangli Fan, Suzhen Wang, Yao Kang, Jianjun Du, and Xiaojun Peng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19281 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

Biodegradable Drug-loaded Hydroxyapatite Nanotherapeutic Agent for Targeted Drug Release in Tumors Wen Sun, Jiangli Fan ⃰, Suzhen Wang, Yao Kang, Jianjun Du, Xiaojun Peng State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 China Email: [email protected]

ABSTRACT: Tumor-targeted drug delivery systems (TTDS) have been increasingly used to improve the therapeutic efficiency of anticancer drugs and reduce their toxic side effects in vivo. Focused on this point, doxorubicin (Dox)-loaded hydroxyapatite nanorods consisting of folic acid (FA) modification (DOX@HAP-FA) were developed for efficient antitumor treatment. The Dox-loaded nanorods were synthesized through in situ coprecipitation and hydrothermal method with DOX template, demonstrating a new procedure for drug loading in hydroxyapatite materials. DOX could be efficiently released from DOX@HAP-FA within 24 h in weakly acidic buffer solution (pH=6.0) due to the degradation of hydroxyapatite nanorods. With the endocytosis under mediation of folate receptors, the nanorods exhibited enhanced cellular uptake, further been degraded, and consequently, the proliferation of targeted cells was inhibited. More importantly, in a tumor-bearing mouse model, DOX@HAP-FA treatment demonstrated excellent tumor growth inhibition. In addition, no apparent side effects were observed during the treatment. These results suggested that DOX@HAP-FA may be a promising nanotherapeutic agent for effective cancer treatment in vivo.

KEY WORDS: biodegradable nanocarriers; hydroxyapatite; targeted release; anticancer drug; folic acid

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1 INTRODUCTION Inefficient therapeutic efficiency and toxic side effects are current two major issues existing in chemotherapy of cancers.1 Nanotechnology plays critical roles in cancer treatment because it provides nanocarriers that can deliver therapeutic agents specifically to the tumor sites.2-4 Nanocarriers, with the size from 10 to 200 nm, can accumulate at the tumor site via enhanced permeability and retention (EPR) effect.5 To enhance the tumor accumulation, nanocarriers can be also functionalized with tumor-targeting group on their surface, which can enable them to target cancer cells through a positive approach.6-13 In this case, when the targeting group binds to the specific receptor expressed on the cell membrane, the payload therapeutic agents would be delivered to the cancer cells without affecting other normal cells. Therefore, the tumor-targeted drug delivery systems (TTDS) can help to improve the specificity of chemotherapy and the therapeutic efficacy, as well as to minimize the undesired side effects of therapeutic agents to healthy tissues.6, 9, 11-13 In recent decades, several inorganic nanomaterials such as gold nanoparticles.14-17 carbon nanotubes,

18-19

and quantum dots

20, 21

have been widely investigated for drug delivery.

Especially, intensive efforts have been made in the construction of TDDS based on mesoporous silica nanoparticles because of their high surface areas and uniformly sized pores for high drug loading efficiency.22-25 However, the poor degradability of inert silica in body may cause long-term toxicity which limits its biomedical applications.26 Several kinds of biodegradable nanocarriers have been reported for drug delivery such as polymeric nanoparticles, organosilica and hydroxyapatite (HAP) nanoparticles. Among them, HAP has been identified as a preferable material to design drug carriers, because HAP is one of the most important constituent of biological tissues, which features excellent biocompatibility and nontoxic to biosystems.27-28 Second, HAP is sensitive to pH and can be degraded into calcium and phosphorous elements under weak acidic conditions. Thus, the complete degradation of the nanoparticles can be achieved through pH mediation.27, 29-31 However, almost all reported 2


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hydroxyapatite nanoparticles carry drugs through surficial adsorption, which could result in drug leakage during blood circulation.27, 28, 30 Drug leakage can cause serious side effects and reduce the bioavailability of the drugs. To develop hydroxyapatite nanocarriers with drug loading inside of the materials would be an approach to solve the above-mentioned problems. To the best of our knowledge, only one example was reported to incorporate bioactive agents (microRNAs) into the HAP nanoparticles.32 However, there was no example that reported the inclusion of small molecule drugs inside the HAP structure. Moreover, hydroxyapatite as drug carriers for in vivo cancer therapy through positive targeting of the tumor has not been reported. Such a design would promote the tumor accumulation of drug carriers and thus enhance their in vivo therapeutic efficiency. To address these issues, we for the first time developed HAP nanoparticles which can encapsulate the anticancer drug inside of the nanostructure. The designed HAP nanocarries can efficiently reduce the drug leakage and improve the availability of the drugs to tumors. Besides, the surface of nanorods was modified with folic acid that can selectively target the tumor site (Scheme 1). DOX is used as a template for HAP nucleation due to the positive charge of the amino group, and then in situ loaded into HAP crystal through the coprecipitation and hydrothermal method. The loading of the anticancer drug into hydroxyapatite structure may enhance the binding force between drug and hydroxyapatite to reduce the drug leakage. Furthermore, surface modification by FA could promote the accumulation of nanorods in tumor sites due to positive tumor targeting. DOX@HAP-FA displayed fast cellular uptake and accumulated in endosome, where DOX can be released due to low pH-induced degradation of hydroxyapatite nanorods. Moreover, the tumor-targeted ability and anticancer effect of DOX@HAP-FA were tested in the fluorescence images of tissues and a tumor-bearing mouse model, demonstrating their potential in clinical applications.

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Scheme 1. Synthesis route of DOX@HAP-FA nanocarrier and the antitumor mechanism of DOX@HAP-FA in cancer cells.

2 MATERIALS AND METHODS 2.1 Materials 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(EDC),

3-

aminopropyltriethoxysilane (APTES), N-hydroxysuccinimide (NHS) and folic acid (FA), were purchased from Energy Chemical (Jiangsu, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT), MEM, DMEM medium and pancreatin were 4


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purchased from HyClone (Logan City, USA). Doxorubicin (DOX) was purchased from a medical company (Jiangsu, China). Calcium nitrate tetrahydrate (Ca(NO3)2·4(H2O)), Diammonium phosphate ((NH4)2HPO4) and hydroxyapatite (HAP) were prepared by State Key Laboratory of Fine Chemicals of DLUT (Dalian, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biological Technology Stock Co., Ltd. (Zhejiang, China). Human breast cancer cells (MCF-7), human hepatoma cell (HepG2) and human liver cells (HL-7702) were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences.

2.2 Cell culture Human breast cancer cells (MCF-7), human hepatoma cell (HepG2) and human liver cells (HL-7702) were applied in cell studies. The cells were incubated in MEM or DMEM medium supplemented with 10% fetal bovine serum (FBS). Then the cells were seeded in 6-well or 96-well flat-bottomed plates and then incubated for 24 h at 37 ºC under 5% CO2 before use (Thermo scientific, USA).

2.3 Animals Nude mice were purchased from Dalian Medical University (Dalian, China). The animal experiments were carried out according to a guidelines approved by the Dalian Medical University Animal Care and Use Committee. The mice bearing tumors were obtained through inoculating ~107 of HepG2 cells in the flank region of each mouse. Tumor volume (V) was calculated as the following formula by measuring length (L) and width (W):13 V

1 L W 2 2

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2.4 Synthesis and characterization of DOX@HAP DOX (30 mg), Ca(NO3)2•4H2O (1.49 g) and (NH4)2HPO4 (0.495 g) were dissolved in 2 mL water, 30 mL ethanol and 2 mL water, respectively. Ammonia was added to Ca(NO3)2 solution to keep the pH at ~10.5. DOX solution was slowly added to the above solution, followed by adding (NH4)2HPO4 to the mixture while stirring. The final volume of the solution was adjusted to 40 mL using ethanol. The suspension was rigorously stirred under ambient conditions for 1 h and then cured in an autoclave at 120 oC for 24 h. The precipitate was obtained through centrifugation and then washed three times by distilled water and ethanol, respectively. Finally, the product DOX@HAP was dried in vacuo at 60 oC overnight and collected for next step. The drug loading capacity was calculated. DOX solution with a concentration of 0.75 mg/mL was prepared before use. After the formation of HAP (0.635 g), DOX concentration of the solution was calculated by measuring the UV absorbance at 480 nm quantified from a standard curve. The concentration of DOX decreased from 0.75 to 0.22 mg/mL. Thus the drug-loading capacity can be obtained through the following equation: Drug loading capacity = Wd/Wr Where Wd represents the amount of DOX that loaded into HAP nanorods; Wr represents the amount of obtained DOX@HAP after preparation. Thus, drug loading capacity can be calculated as follows:

(0.75-0.10) mg/mL×40

mL/0.635 g = 41 mg g-1.

2.5 Synthesis and characterization of DOX@HAP-NH2 100 mg 3-aminopropyltriethoxysilane (APTES) was added to a mixed solvent (ethanol/H2O = 27 mL/3 mL), followed by stirring for 2 h. Then 300 mg DOX@HAP was added and sonicated for 3 min to allow the HAP to be well dispersed in the mixture. After stirring for 2 6


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h, the pH of the solution was adjusted to 10 using ammonia and continued stirring for 3 h at room temperature (r. t.). The obtained precipitate was washed by absolute ethanol for three times to remove the excess APTES. The intermediate product (DOX@HAP-NH2) was obtained through centrifugation and vacuum drying.

2.6 Preparation and characterization of DOX@HAP-FA The activation of FA was achieved from the reaction of FA (1.1×10-4 mmol, 50 mg) and EDC (1.1×10-4 mmol, 22 mg) in deionized water (70 mL) for half an hour at r.t.. After that, NHS (2.75×10-4 mmol, 32 mg) was added to the above solution and kept stirring for another 6 h. The NHS activated FA further reacted with DOX@HAP-NH2 (100 mg) in pH 7.4 phosphate buffer for 3 h. The resultants DOX@HAP-FA were washed with deionized water and ethanol to remove the unreacted chemicals through centrifugation. The final nanoparticles were obtained through centrifugation and vacuum drying. In order to determine the amount of surface-grafted FA, DOX@HAP-FA (30 mg) was incubated in acid condition (pH = 5) for two days, which can lead to hydrolysis of the nanorods. The concentration of FA in the solution can be determined through measuring the UV absorbance at 340 nm quantified from a standard curve. Thus FA-grafting capacity can be obtained through the following equation: FA-grafting capacity = Wf/Wd Where Wf represents the amount of FA that grafted to the nanorods; Wd represents the amount of DOX@HAP-FA. Accordingly, FA-grafting capacity was calculated as 11.6 mg g-1.

2.7 In vitro drug doping and drug release profile The FA conjugated hydroxyapatite nanoparticles were used as nanocarriers for anticancer drug DOX. Loading content and release behavior of DOX@HAP-FA were detected by UVvis spectroscopy. Phosphate buffer medium with different pH values (pH 7.4, 6.0 and 5.5) 7



were prepared to mimic the pH environment of blood, cancer cells and endosomes. DOX@HAP-FA were incubated in the above solutions at 37 oC with gentle agitation. The drug release was monitored by UV-vis spectroscopy and repeated for three times (n=3). The curve of UV-vis spectroscopy reflected the DOX release behavior from DOX@HAP-FA nanocarriers.

2.8 Cell viability All mentioned cell viability was assessed by standard MTT (3-(4, 5)-dimethylthiahiazo (-2yl)-3, 5-diphenytetrazoliumromide) assay in different cell lines. HL-7702, MCF-7 and HepG2 cells were seeded in 96-well plates (Nunc, Denmark) at a density of 1×105 cells/mL in 100 μL medium containing 10 % FBS. After 24 h of cell attachment, the plates were washed by 100 μL/well PBS. The cells were then cultured in medium with DOX@HAP (10, 20, 30, 40, 50 mg mL-1) or DOX@HAP-FA (10, 20, 30, 40, 50 mg mL-1) for 24 h. Cells incubated without any treatment were used as the control experiment. After different treatment, MTT PBS solution (10 μL, 5mg/mL) was added to each well, followed by incubation at 37 ºC for another 4 h in a humidified incubator with 5% CO2. The medium was then carefully removed, and the purple crystals were lysed in 200 μL DMSO. Optical intensity was obtained from a microplate reader (Thermo Fisher Scientific) at 570 nm with subtraction of the absorbance of the cell-free blank volume at 490 nm.

2.9 Fluorescence imaging of live cells stained with DOX@HAP-FA DOX@HAP-FA was individually added to normal and cancer cells. Cells were incubated for 30 min at 37 ºC under 5 % CO2 and then washed with PBS buffer for three times. Fluorescence images were collected by an OLYMPUSFV-1000 inverted fluorescence confocal microscope, using a 60×objective lens. DOX@HAP-FA (red channel) was excited at 8


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488 nm, and the emission spectra were collected at 540-600 nm.

2.10 Flow cytometry Flow cytometry was carried out to quantify the fluorescence intensity from DOX@HAP-FA incubated HL-7702 and HepG2 cells, which demonstrate low FR and higher FR expression, respectively. The measurement can further prove the specific cellular uptake of DOX@HAPFA through FR-mediated endocytosis. Generally, cells (5×104) were seeded into 6-well plates one day before use. DOX@HAP-FA nanorods were added to the cell solution with the final concentration of 0.5 mg mL-1. Cells were incubated for 2h and then washed twice with PBS solution before flow cytometry (Attune NxT) analysis.

2.11 In vivo antitumor study The antitumor effect of DOX@HAP-FA was conducted on tumor-bearing mice. The mice were divided into three groups when the tumor volume reached to ~ 50 mm3: (1) DOX@HAP at 5 mg DOX/kg; (2) free DOX at 5 mg DOX/kg; (3) DOX@HAP-FA at 5 mg DOX/kg. The mice were treated through paracancerous injection with DOX@HAP, DOX, or DOX@HAPFA. The tumor volume of each mouse was measured and recorded over a period of 21 days. The body weight was also recorded during the treatment. At day 21, the mice were sacrificed, and tumors were isolated.

3 RESULTS AND DISCUSSION 3.1 Characterization of DOX@HAP-FA To study the structure of HAP, N2 adsorption–desorption isotherms of HAP was first tested. The Brunner-Emmet-Teller (BET) pore volume and Barrett-Joyner-Halenda (BJH) pore diameter were not detected, indicating that there was almost no mesopores in the structure 9



(Figure 1). SU8220 EDS measurement confirmed that HAP contained oxygen (O), phosphorus (P), and calcium (Ca) elements, and the three elements were uniformly distributed in the nanoparticles (Figure S1, S2 Supporting Information). Additionally, the ratio of Ca/P of the nanoparticle was 1.42 which is among the reported range of hydroxyapatite (1.33-1.67) [33] (Tab. S1, Supporting Information). These results demonstrated that the hydroxyapatite nanoparticles were successfully synthesized.

Figure 1. (a) N2 adsorption-desorption isotherms, and (b) pore size distributions of HAP nanoparticles.

DOX@HAP-FA were synthesized through coprecipitation and hydrothermal method. The structural properties of DOX@HAP-FA were well analyzed by a series of characterization techniques. TEM and SEM images revealed that DOX@HAP-FA features a nanorod morphology, with anisotropy and uniform size distribution (Figure 2a, b). The average length, diameter and height of nanorods were 140, 28 and 22 nm measured from the SEM image (Figure 2b). In addition, the nanorods were also studied by dynamic light scattering (DLS), which showed a narrow size distribution with an average diameter of 141 nm (Figure S3). Anisotropic particles like nanorods have been demonstrated to have better tumor targeting property than spherical nanoparticles due to different motion characteristics when injected into the blood vessels.27 Furthermore, unlike spherical nanoparticles which move along the 10


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blood vessel parallelly, the anisotropic particles exhibit more complex motions including rolling and tumbling in the blood flow.27 The movements make the particles easy to touch the vessel walls, which can facilitate the passive tumor targeting through EPR effect.34-35 Thus DOX@HAP-FA may have good tumor-targeting effect in vivo due to its anisotropic nanostructure. The FT-IR spectra of HAP, DOX@HAP, DOX@HAP-NH2 and DOX@HAP-FA are shown in Figure 2c. The intense absorption peak at 1020 cm-1 belongs to the stretching vibration of the phosphate (PO43-) groups of the nanorods. While the peaks located at 565 and 610 cm-1 are assigned to the bending vibration of phosphate (PO43-) groups. The broad absorption band centered at 3400 cm-1 is attributed to OH group, which is formed from hydrogen bonded OH group with phosphate group and can also from the interaction between OH groups and water vapour. The reduction of the transmittance at 1384 cm−1 of HAP after DOX loading was indicative of secondary bonding between δ O–H···O (of DOX) and –CH2– of HAP (Figure 2c). The peak located at 1580 cm-1 is ascribed to the N–H stretching vibrations, suggesting that the amino groups were successfully grafted onto the surface of DOX@HAP nanorods. The peak of DOX@HAP-FA located at 1685 cm-1 and the peaks at 1610 and 1505 cm-1 belonged to stretching and in-plane bending vibration of the carbonyl group in newly formed amide, respectively. Clearly, FA groups were successfully introduced to the surface of the nanorods. Furthermore, as shown in Figure 2d, the zeta potential value of HAP and DOXdoping HAP (DOX@HAP) exhibited low positive values (0.333 and 0.425). While the potential value of aminated DOX@HAP (DOX@HAP-NH2) greatly increased to 26.80, due to the protonation of the surface grafted amine groups. Finally, after the surface of DOX@HAP-NH2 was modified by FA, the potential value of DOX@HAP-FA decreased sharply to 0.452 again. The surface charge changes in each step demonstrated the successful synthesis of DOX@HAP-FA. In addition, the wide-angle XRD analysis of DOX@HAP-FA was also studied (Figure 2e). Three strong specific diffraction peaks (211), (112), (300) at 2θ 11



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= 31.9, 32.2 and 32.8 could be detected in XRD spectrum. Additionally, other peaks of the nanorods are also corresponding to the pure hydroxyapatite (Figure S4, supporting information), suggesting that the nanorods kept intact after drug-loading and surface modifications. The relative crystallinity of DOX@HAP-NH2 was evaluated by comparing the strongest peak (211) of the HA, which decreased to 52 % due to the loading of DOX (Figure S4, supporting information). Besides, after the addition of DOX, the 2θ peak position of (0 0 2) shift from 25.64 to 25.60o. Thus, the plane spacing of (0 0 2) is decreased from 3.468 to 3.476 Å, which demonstrated that DOX adsorbed on the (0 0 2) plane of HA (Figure S4, supporting information).

[36]

Moreover, we calculated the Brunner-Emmet-Teller (BET)

surface areas of DOX@HAP and DOX@HAP-FA based on BET theory as ~34 m2 g-1, and ~31 m2 g-1, respectively. (Figure S5, supporting information).

[37]

So after drug-loading, the

BET specific surface area was the same as that of the HAP nanorods (~34 m2 g-1), suggesting that the drug-loading did not take place on the surface of the nanorods. While, the BET surface decreased from ~34 m2 g-1 to ~31 m2 g-1 due to the surface modification of FA.

12


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Figure 2. (a) TEM image of DOX@HAP-FA. Scale bar: 100 nm. (b) SEM image of DOX@HAP-FA. Scale bar: 150 nm. FT-IR spectra (c) and (d) zeta potential measured at each step of the preparation process. (e) X-diffraction spectrum (XRD) of DOX@HAP-FA (JCPDS 09-0432).

3.2 Spectral properties of DOX@HAP-FA The UV-Vis absorption spectra of folic acid and DOX@HAP-FA are shown in Figure 3a. Folic acid displayed a strong absorbance at ~280 nm, and a further strong peak at ~370 nm (Figure 3a, back curve). The two typical absorption peaks were also observed in the absorption spectrum of DOX@HAP-FA, indicating the successful conjugation of folic acid on the surface of HAP nanorods (Figure 3a, red curve). Fluorescence spectra were used to verify that the drug was loaded inside of the HAP. Free DOX exhibited a strong emission peak centered at ~590 nm upon the excitation of 480 nm (Figure 3b, black curve). However, no emission peak could be observed from DOX@HAP-FA after drug loading (Figure 3b, red curve). The fluorescence quenching is ascribed to the energy transfer between the drug and HAP materials. However, after hydrolysis of the nanorods, the fluorescence spectrum 13



appeared again and was in accordance with free DOX (Figure S6, supporting information). These results demonstrated that DOX was loaded safely inside of the nanorods.

Figure 3. (a) Absorption spectra of FA and DOX@HAP-FA in PBS (pH = 7.4). (b) Fluorescence emission (λex = 480 nm) of DOX and DOX@HAP-FA in PBS (pH = 7.4).

3.3 In vitro drug doping and drug release profile The drug doping capability of DOX@HAP was calculated to be 41 mg g-1. After two-step surface modification the doping capability of DOX@HAP-NH2 and DOX@HAP-FA decreased to 37.7 mg g-1 and 33.2 mg g-1, which was calculated by measuring the released DOX in the reaction solution. The final nanorods can be well dispersed in aqueous solution and the size was nearly no change even after 14 day’s incubation (Figure S7, supporting information). Thus DOX@HAP-FA was used for in vitro drug release experiment (Figure 4). The experiment was carried out under the circumstances of sink condition at 37 C. As to mimic the biological conditions, PBS solutions with different pH values were prepared. A pH 7.4 solution was to simulate the blood circulation, while the solution of pH 6 and 5.5 were for tumor cells and endosome, respectively. 38 The DOX release percentage from the nanorods in pH 7.4 PBS was only around 16 % after 24 h incubation, demonstrating that DOX@HAP-FA can avoid the drug burst release from the nanocarriers and thus enhance the availability of the drug to tumors. On the contrary, DOX release from the nanorods increased dramatically to 14


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38 % in 2.5 hour and then gradually increased to 74 % and 82 % at pH 6 and pH 5.5 respectively within the same time period. The efficient drug release revealed that DOX@HAP-FA could release DOX under weakly acidic conditions. This was because HAP was degraded to calcium and phosphate ions in acid conditions, which weakened the interaction between HAP nanoparticles and DOX.39-41 In addition, due to the pH-sensitive properties of hydroxyapatite materials, the interaction between calcium and protons could also reduce the adsorption capacity of HAP to DOX.40-41 To prove the degradable properties of the nanorods, we investigated the degradation of DOX@HAP-FA by monitoring the Ca2+ concentration in different pH solutions (Figure S8, supporting information). In neutral condition (pH 7.4), nearly no Ca2+ can be detected from the solution, which demonstrated that the nanorods didn’t show any degradation. While in acidic conditions (pH 6.5, 6, 5.5), Ca2+ concentration of the solutions increased dramatically during incubation for 24 h. Besides, the Ca2+ concentration in solutions with pH 6 and pH 5.5 was higher than that of pH 6.5. These results suggested acidic conditions result in degradation of the HAP nanorods and lower pH would make the degradation of nanorods more thoroughly.

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Figure 4. Release profiles of DOX from HAP in different pH buffers (n=3). Error values are shown in Tab. S2 in the supporting information.

3.4 Cytotoxicity assay In order to verify that the FA-modified nanorods can target folate receptor (FR)overexpressing tumor cells, in vitro cytotoxicity of DOX@HAP-FA against normal cells (HL7702, FR(-)) and cancer cells (MCF-7, FR(-); HepG2, FR(+)) were conducted by using MTT assay. The viability of HL-7702 and MCF-7 cells remained at ~80 % with increasing the nanorod concentrations (Figure 5a). However, the viability of HepG2 cells gradually decreased with increase the concentration of DOX@HAP-FA nanorods. The results clearly demonstrated that the enhanced cytotoxicity of DOX@HAP-FA to HepG2 cells was concentration-dependent and the presence of folic acid made DOX@HAP-FA display stronger cellular inhibition towards FR-overexpressing tumor cells. To further prove the specific tumor targeting of FA, the viability of HepG2 cells treated by bare HAP and nanorods with or without FA modification were also tested (Figure 5b). As expected, bare HAP demonstrated little toxicity. While both DOX@HAP and DOX@HAP-FA can result in cell death due to the intracellular drug release. Notably, nanorods consisting of FA displayed higher toxicity than DOX@HAP, further demonstrating that the modification of FA could 16


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enhance the cellular uptake of the nanoparticles.

Figure 5. (a) Cell viability of HL-7702, MCF-7 and HepG2 after treatment with DOX@HAP-FA at different concentrations. (b) Cell viability of HepG2 after treatment with HAP, DOX@HAP or DOX@HAP-FA at different concentrations. Error values are shown in Tab. S3, S4 in the supporting information.

3.5 Cellular uptake of DOX@HAP-FA The cellular uptake of DOX@HAP-FA by normal cells (HL-7702) and tumor cells (MCF-7, HepG2) were evaluated by confocal laser fluorescence imaging. After 2 h-incubation, the nanorods only adhered on the cell membrane and seldom internalized into HL-7702 and MCF-7 cells with low FR-expression (Figure 6a, b). On the contrary, DOX@HAP-FA demonstrated fast cellular uptake by HepG2 cells; strong fluorescence can be observed from the whole cytoplasm after 2 h incubation (Figure 6a, b). The results showed that DOX@HAPFA could specifically recognize FR-overexpressing tumor cells and fast internalized into the cells. Flow cytometry measurement is able to count cell numbers and record fluorescent intensities at the same time. Thus, flow cytometry analysis was also used to quantify the cellular uptake of nanorods in different cell lines. The flow cytometric experiment demonstrated a remarkable cellular uptake of nanorods to HepG2 compared to MCF-7 (Figure S9, Supporting Information). The result was in accordance with confocal laser fluorescence images and further proved that the nanorods showed efficient cellular uptake to FR17



overexpressing cancer cells.

Figure 6. (a) Confocal laser fluorescence images of HL-7702, MCF-7 and HepG2 cells after incubation with DOX@HAP-FA for 0.5 h, 1 h and 2 h. (scale bar: 20 µm). (b) Average fluorescence intensity obtained from fluorescence images after 2 h incubation with DOX@HAP-FA.

In vitro tissue imaging experiments were also conducted. Tumor and normal tissues including kidney, heart, liver, lung, and spleen and tumor tissue were immersed into the solution of DOX@HAP-FA (50 mg/L) for 6 h, followed by raised with water for three times. Fluorescence images of tumor and different organs were obtained under an in vivo imaging system. Although some fluorescence was detected from healthy organs including heart, liver, kidney and spleen, the fluorescence of tumor was much stronger (Figure 7). This phenomenon 18


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is in accordance with the cell imaging experiment. The HAP nanoparticles can also be uptake by normal cells (HL-7702), but cancer cells (HepG2) demonstrated much more efficient cellular uptake. However, even a few nanoparticles were uptake by healthy cells, HAP nanoparticles are not toxic to the cells due to the neutral pH environment where the nanoparticles are not degraded. Tissue penetration of nanoparticles plays an important role in anticancer therapy, especially in solid tumors. We further performed tissue depth imaging to prove their tumor penetration ability and validate the targeted effect of FA (Figure 7). The penetration depth of DOX@HAP-FA to normal tissue including heart, lung and kidney were about 20 μm, while the depth to tumor tissue was 60 μm. Thus, the FA-modified nanorods demonstrate better permeability towards tumor tissue due to the specific cancer cell targeting.

Figure 7. Ex vivo images of tumor and other organs after 6 h incubation with 50 mg/L DOX@HAPFA in vitro (left). Tissue depth images of heart (a), lung (b), kidney (c) and tumor (d) after 6 h incubation with 50 mg/L DOX@HAP-FA in vitro (right).

Overall, DOX@HAP-FA can preferentially target the cancer cells with high FR expression through the active targeting ability of FA. In addition, the nanrods showed good tumor penetration in tissue imaging. These results demonstrated that DOX@HAP-FA is suited for further in vivo antitumor therapy.

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3.6 In vivo antitumor efficacy test The antitumor efficacy of DOX@HAP-FA was determined in vivo. To this end, we implanted HepG2 in the left armpit of nude mice. After tumor establishment, the mice were given continuous therapy through injection with free DOX, DOX@HAP, or DOX@HAP-FA. Relative tumor volume was recorded during three weeks (Figure 8a). In the experiment, the solid tumor was induced under the skin which was visible to naked eyes. Thus paracancerous injection was chosen for the treatment which is the most direct and easiest way to prove the tumor targeting ability of the FA-modified nanorods. Compared with free DOX group, the group treated with DOX@HAP showed a moderate tumor inhibition capability due to the release of DOX in tumor sites by DOX@HAP. However, it is notable that DOX@HAP-FA brought about the strongest effect on suppressing the tumor growth, which validated that FA modification enhanced the tumor targeting and thereby enabled the reinforcement on the antitumor efficacy. At Day 21, the average tumor volume in DOX and DOX@HAP groups reached to 10 and 5-fold, respectively. While only 2.5-fold increase in average tumor volume was observed in DOX@HAP-FA group. To prove the importance of targeting group in vivo, we investigated the distributions of DOX@HAP-FA in different organs including heart, kidney, liver, spleen, and lung (Figure S10, supporting information). Since the fluorescence of DOX cannot be directly detected in vivo, we isolated the organs and measured their fluorescence intensity through a fluorescence imaging system. After injection of the nanorods for 6h, strong fluorescence was observed from the tumor, while only very week fluorescence can be detected from the other organs. The results demonstrated that DOX@HAP-FA can efficiently accumulate in the tumor site due to the surface-modified targeting groups. The biosafety of DOX@HAP-FA was also evaluated. The body weights of the mice were monitored for 21 days (Figure 8b). Our treatment did not decrease the body weights of the mice, indicating that the injection of DOX@HAP and DOX@HAP-FA had minimal side 20


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effects on the mice. On the contrary, apparent loss in body weight was observed in free DOX group, which is mainly caused by the side effects of the drug. In addition, hemolysis analysis was conducted to study the blood compatibility of DOX@HAP-FA nanorods (Figure S11). Once red cells were put into pure water, the cells were destroyed immediately. However, no hemolytic effect can be observed from red cells after incubation with the nanorods. The result demonstrated good compatibility of the nanorods to blood.

Figure 8. (a) Relative tumor volume during different treatments (n=3). Insert shows the images of tumors from each group (from left to right: free DOX, DOX@HAP, and DOX@HAP-FA). Error values are shown in Tab. S5 in the supporting information. (b) Relative body weights of tumorbearing mice during different treatments (n=3).

4 CONCLUSION In this study, FA modified drug-nanocarriers, DOX@HAP-FA nanorods, were developed for efficient antitumor therapy in vivo. The nanocarriers were synthesized through coprecipitation with DOX, suggesting a new method to load drug in hydroxyapatite materials. DOX@HAPFA exhibited good stability in neutral solution, but effective pH-induced drug release under low pH conditions. With the surface-modified FA, DOX@HAP-FA could specific target FRoverexpressing tumor cells through the positive targeting. The more efficient intracellular delivery of DOX by DOX@HAP-FA resulted in higher therapeutic effect in FR21



overexpressing tumor cells. In vivo antitumor investigation on tumor-bearing mouse mode confirmed the enhanced tumor-targeting and excellent anticancer effect of DOX@HAP-FA. Moreover, the nanorods did not cause any noticeable side effects to mice during the treatment. Therefore, DOX@HAP-FA is a promising nanotherapeutic agent for in vivo antitumor therapy and may have applications in future clinics.

5 ASSOCIATED CONTENT Supplementary information is available: EDX analysis, additional spectroscopic, XRD analysis, nanoparticle stability, nanoparticle degradation, cell viability, and biodistribution analysis.

6 ACKNOWLEDGMENTS This work was financially supported by National Science Foundation of China (21576037, 21422601, 21676047, 21421005), NSFC-Liaoning United Fund (U1608222).

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Biodegradable Drug-Loaded Hydroxyapatite ... - ACS Publications

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