Alginate CoreâShellâCorona Nanoparticles

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Tri-functional Fe3O4/CaP/Alginate Core-ShellCorona Nanoparticles for Magnetically Guiding, pHResponsive, and Chemically Targeting Chemotherapy Yu-Pu Wang, Yu-Te Liao, Chia-Hung Liu, Jiashing Yu, Yusuke Yamauchi, Md. Shahriar A Hossain, and Kevin C.-W. Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00230 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Biomaterials Science & Engineering

Tri-functional Fe3O4/CaP/Alginate Core-Shell-Corona Nanoparticles for Magnetically Guided, pH-Responsive, and Chemically Targeted Chemotherapy

Yu-Pu Wang,a# Yu-Te Liao,a# Chia-Hung Liu,b Jiashing Yu,a Hatem R. Alamri,c Zeid A. Alothman,d Md. Shahriar A. Hossain,e,f Yusuke Yamauchi,e,f and Kevin C.-W. Wu*a,g

a

Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan.

b

Department of Urology, Taipei Medical University - Shuang Ho Hospital, No. 291, Jhongjheng Rd., Jhonghe Dist., New Taipei City 23561, Taiwan.

c

Physics Department, Jamoum University College, Umm Al-Qura University, Makkah 21955, Saudi Arabia.

d

Advanced Materials Research Chair, Chemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.

e

Australian Institute for Innovative Materials (AIIM), University of Wollongong, Squires Way, North Wollongong, NSW 2500, Australia.

f

International Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

g

Division of Medical Engineering Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan.

#

Equal contributions by the first two authors

Fax: +886-2-33663040; Tel: +886-2-33663064; E-mail: [email protected] 1 ACS Paragon Plus Environment


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Abstract Chemotherapy of bladder cancer has limited efficacy due to the short retention time of drugs in the bladder during therapy. In this research, nanoparticles (NPs) with a new core/shell/corona nanostructure have been synthesized, consisting of iron oxide (Fe3O4) as the core to providing magnetic properties, drug (doxorubicin) loaded calcium phosphate (CaP) as the shell for pH-responsive release, and arginylglycylaspartic acid (RGD)-containing peptide functionalized alginate as the corona for cell targeting (with the composite denoted as RGD-Fe3O4/CaP/Alg NPs). We have optimized the reaction conditions to obtain RGD-Fe3O4/CaP/Alg NPs with high biocompatibility, and suitable particle size, surface functionality, and drug loading/release behavior. The results indicate that the RGD-Fe3O4/CaP/Alg NPs exhibit enhanced chemotherapy efficacy towards T24 bladder cancer cells, owing to successful magnetic guidance, pH-responsive release, and improved cellular uptake, which give these NPs great potential as therapeutic agents for future in vivo drug delivery systems.

Keywords: core-shell-corona nanoparticles, bladder cancer, controlled release, magnetic guidance, targeting.

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1. Introduction Bladder cancer, accounting for 3.2% of all cancers, has been more and more attention all over the world.1 Although about 70% of bladder cancers belong to non-muscle invasive types, mainly managed with an endoscopic resection procedure (ERP), over 50% of non-muscle invasive tumors come back a few years later after surgery, resulting in metastasis of the tumor.2 After the ERP, there are some cancer cells that still remain in the patient’s bladder. The conventional treatment after the ERP is systemic administration of drugs, but this chemotherapy is usually ineffective due to the complications of the human body. Most of the drug molecules are decomposed by enzymes, and only a small fraction of the drug can reach the tumor site.3 Intravesical therapy, involving direct administration of drugs, is a popular therapeutic strategy in bladder cancer treatment after ERP.4 The drug molecules are directly injected into the bladder through a catheter, thus providing maximal delivery efficacy and minimal systemic side effects.3 Bacillus Calmette-Guerin (BCG) is commonly used in the intravesical therapy,5

but

severe

side

effects

happen

occasionally.

Alternative

chemotherapeutic agents, such as doxorubicin, mitomycin C, and paclitaxel, are also used in intravesical therapy.6-8 Even when these drugs can be delivered via intravesical treatment, however, the probability that the drug will reach the tumor site in the bladder is still low.9 This is because it is necessary to rotate the patients treated with intravesical injection during treatment to ensure that the drugs can be efficiently taken up by the cancer cells. The whole treatment usually lasts for less than 30 min because the patients cannot tolerate the pain, and the drug solution instilled in the bladder is exhausted by the periodical voiding of urine. Consequently, frequent dosage is required to maintain the concentration of drug 3 ACS Paragon Plus Environment


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solution in the bladder, which causes several side effects such as irritation during voiding, bladder fibrosis, and infections.10 The efficacy of the intravesical injection depends on the retention time of drug molecules inside the bladder and on the extent of the interaction between the drug and the urinary bladder wall.3 So far, several physical and chemical methods have been utilized to enhance the attachment of chemotherapeutic drugs to the bladder wall.11-14 Electromotive drug administration conjugated with intravesical injection improved the penetration rate of drugs into the bladder wall.12 The electric field needs to be strictly monitored, however, to prevent irreversible damage to the bladder tissue. Some chemical penetration enhancers, such as dimethyl sulfoxide and protamine sulfate, are also widely used to react with glycosaminoglycans, which are anti-adherent on the bladder wall, thus enhancing the penetration of the drug.11 The drawbacks of this method are side effects such as incontinence, pelvic pain, and urethral burring if the chemical enhancers are not regulated properly.15-16 Carrier-based drug delivery systems (DDS) applied in intravesical injection have become popular because of the advantages of high drug-loading capacity, low side effects, and high penetration of the drug.17-19 Frangos et al. encapsulated interferon alpha (IFN-α) in liposomes and delivered it to human bladder carcinoma cell line 253J.20 The anti-proliferative effect of IFN-α increased when 253J was treated with liposome-IFN-α complex. Even when the cell line was changed to a drug resistant subline, the effect of IFN-α was still more obvious than for the free drug. Lu et al. delivered the hydrophobic drug paclitaxel (PTX) to bladder cancer by encapsulating the drug in gelatin nanoparticles.21 Their results indicated that the concentration of PTX delivered by gelatin nanoparticles in the tissue was three 4 ACS Paragon Plus Environment

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times higher than for free PTX. Erdogar et al. encapsulated mitomycin C in chitosan coated poly-ε-caprolactone nanoparticles (CS-PCL) for DDS to the bladder.1 They found out that the group of mice treated with mitomycin C-encapsulated CS-PCL had the highest survival rate among all the other groups. Despite these interesting results on carrier-based drug delivery systems, however, the main challenge for intravesical injection is how to accelerate cellular uptake in such a short retention time when drug molecules are injected into the three-dimensional (3D) space of bladder. Magnetic iron-oxide based nanoparticles can be the solution to this problem because they can be easily guided by an external magnetic field.9,22 Besides magnetic guiding, iron-oxide based nanoparticles could be also applied for hyperthermia and mechanical damage.23-25 Cheng et al. fabricated iron oxide nanoparticles with the up-conversion property,26 and their results indicated that the uptake of nanoparticles guided by external magnetic field was enhanced up to 8-fold. Kim et al. utilized poly(lactide-co-glycolide) (PLGA) as template to encapsulate quantum dots, doxorubicin, and iron oxide.27 They asserted that they had achieved successful in-vivo cancer-targeting, magnetic resonance imaging (MRI) of the cancer, and optical imaging by using their multifunctional polymer nanoparticles. In the case of bladder cancer, Leakakos et al. used magnetic targeted carriers (MTCs) to deliver doxorubicin intravesically,9 and they found that the distribution of MTCs was predominantly at the targeted site. Zhang et al. synthesized magnetic chitosan hydrogel to deliver BCG to the bladder.28 They claimed that compared with traditional BCG therapy, BCG delivered by chitosan gel induced a stronger Th1 immune response and revealed higher antitumor efficacy. These pioneering reports, above all, indicate the usefulness of magnetic 5 ACS Paragon Plus Environment


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guidance in intravesical injection for bladder cancer therapy. It would be ideal, however, if one could combine the magnetic guidance with other properties such as pH-responsive release and targeting to maximize the advantages of carrier-based DDS. In this research, we propose a tri-functional nanoparticle with the capabilities of magnetic guidance, cancer cell-targeting, and pH-responsive release for bladder cancer therapy (Scheme 1). Iron oxide nanoparticles were first synthesized in alginate solution by co-precipitation of ferric ion.29 The network of alginate polymer could achieve a good suspension of iron oxide nanoparticles in the solution. Calcium phosphate as a pH-response shell was then formed on the alginate through the pre-gel method.30 Doxorubicin was loaded between the iron oxide core and the calcium phosphate shell. The abundant carboxylate groups on alginate polymer allowed further functionalization with a cancer cell-targeting motif (i.e. an RGD-containing peptide, c(RGDfK)K3) that can target the αvβ3 integrin of bladder cancer cells (i.e. T24). The proposed intravesical drug delivery system consists of iron oxide, calcium phosphate, and alginate as core, shell, and corona (denoted as Fe3O4/CaP/Alg), respectively. The nanoparticles would be guided to the location of tumor by external magnetic field followed by passive targeting between RGD-based peptide and αvβ3 integrin. The pH-responsive shell, CaP could prevent drugs from leaking during intravesical administration and release the drugs in relatively acidic environment such as tumor tissue. We have confirmed that the Fe3O4 core provides the magnetic guidance property, the CaP shell exhibits a pH-responsive release property, and the alginate corona can link with RGD peptides for targeting ability. When bladder cancer cells (i.e. T24) are treated with doxorubicin loaded Fe3O4/CaP/Alg in our system, great enhancement 6 ACS Paragon Plus Environment

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of anticancer efficacy is realized.

Scheme 1. (a) Illustration of the synthesis of iron oxide/calcium phosphate/alginate core-shell-corona nanoparticles with an RGD-containing peptide as the cancer cell targeting ligand (with the composite denoted as RGD-Fe3O4/CaP/Alg). (b) Illustration of the magnetically guided, pH-responsive, and chemically targeted chemotherapy using the synthesized RGD-Fe3O4/CaP/Alg.

2. Materials and Methods 2.1. Chemicals Iron(II) chloride tetrahydrate (FeCl2·4H2O, 98%) was purchased from Alfa Aesar.

Iron(III)

chloride

1-(3-dimethylaminopropyl)-3-ethylcarbodimmide (NHS),

hexahydrate (EDC),

(FeCl3·6H2O), N-hydroxysuccinimide

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

potassium ferrocyanide (K4[Fe(CN)6]·3H2O), 10% neutral buffered formalin, nuclear fast red counterstain, and sodium alginate were purchased from Sigma-Aldrich. Alginic acid sodium salt, calcium chloride, doxorubicin hydrochloride, and sodium phosphate dibasic were purchased from Sigma-Aldrich without further purification. Cyclic RGD peptide (c(RGDfK)K3) was provided by Kelowna (Taiwan). Phosphate buffered saline (PBS, 10x), fetal bovine serum (FBS), sodium bicarbonate (7.5 wt%), L-glutamine (200 mM in saline), tripsin-ethylenediamine tetraacetic acid (EDTA) 10×,



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and Pen-Strep Sol. (penicillin 10000 units mL-1 and streptomycin 10 mg mL-1) were provided by Biological Industries. 2.2. Synthesis of alginate covered Fe3O4 containing calcium phosphate nanoparticles (Fe3O4/CaP/Alg NPs) 10 mg of sodium alginate was dissolved in 55 mL of deionized (DI) water. 98 mg of ferric(II) chloride dihydrate and 169 mg of ferric(III) chloric tetrahydrate dissolved in 10 mL of DI water were injected respectively into the alginate containing solution at the rate of 0.5 mL/min. After the previous solution was vigorously stirred for 1 hour, the pH value of the solution was adjusted to 10 by ammonia solution (1.5 N) and stirred for a further 30 min. The as-synthesized Fe3O4/Alg NPs were collected by magnet and washed 2 times with DI water. The suspension was diluted to 0.8 mg/mL for storage. 1.5 mL of calcium chloride solution (0.06 M) was injected into 25 mL of Fe3O4/Alg (0.8 mg/mL) solution at the rate of 0.25 mL/min. After stirring for 1 h, 1.2 mL of sodium phosphate dibasic (0.045 M) was injected into the solution at the rate of 0.25 mL/min, followed by stirring for a further 30 min. The brown suspension, alginate covered Fe3O4 containing calcium phosphate nanoparticles, was collected by magnet and washed three times. In order to load doxorubicin (Dox) into the space between core and shell, 1 mL of Dox (1 mg/mL) was injected into 25 mL of Fe3O4/Alg (0.8 mg/mL) solution at the rate of 0.25 mL/min before the injection of sodium phosphate dibasic. After the Dox containing solution was injected into the Fe3O4/Alg solution, the following processes were the same as with Fe3O4/CaP/Alg. The Dox containing NPs were denoted as Dox/Fe3O4/CaP/Alg. The supernatant of the Fe3O4/CaP/Alg, including the washing solution, was collected and analyzed by photoluminescence (PL) to calculate the amount of encapsulation and the efficiency. 8 ACS Paragon Plus Environment

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The encapsulation amount and efficiency were calculated based on the two following formulas:

Encapsulation amount μg⁄mg = Encapsulation efficiency % =

Dox μg in feed − Dox in supernatant μg Fe O /CaP/Alg mg

Dox μg in feed − Dox in supernatant μg Dox μgin feed

2.3. Functionalization of Fe3O4/CaP/Alg and Fe3O4/CaP/Alg with cyclic RGD peptide (c(RGDfK)K3 10 mg of Fe3O4/CaP/Alg was suspended in 8 mL of phosphate buffered saline (PBS, pH = 7.4). 49.8 mg of EDC, and 74.8 mg of NHS were first dissolved in 2 mL of PBS (pH = 7.4) and then added into the previous solution and stirred for 30 min. The NPs were collected by magnet, and the supernatant containing unreacted EDC and NHS was removed. The NP containing slurry was re-suspended in a 10 mL solution of RGD peptide (0.1 mg/mL) in PBS (pH = 8.5) and stirred for 4 h. The NPs and supernatant were separated by magnet again and washed with PBS (pH = 12). The NP containing slurry was re-suspended in 5 mL of PBS (pH=12) and stirred for 30 min to reduce the activated carboxylic groups. Finally, the NPs were washed with DI water 2 times and stored in DI water. 2.4. Characterization The hydrodynamic diameter and zeta potential of the synthesized Fe3O4/Alg and Fe3O4/CaP/Alg NPs were measured on a Zetasizer Nano ZS system (Malvern Instruments Ltd., UK) at 25 °C with DI H2O as the solvent. Samples were sonicated for 1 h before measuring. The average particle size was evaluated based on dynamic light scattering (DLS). The morphology and the core-shell nature of the samples were observed using a scanning electron microscope (SEM; NovaTM Nano SEM) and a transmission electron microscope (TEM; JEOL JEM-1200EX II). The Fe3O4/Alg and Fe3O4/CaP/Alg NPs suspension were sonicated for 1 h, dropped onto copper grids 9 ACS Paragon Plus Environment


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(200 mesh, carbon-coated), dried under vacuum, and subjected to Pt coating before TEM observation. Functional groups of the samples were examined with Fourier transform infrared spectroscopy (FTIR; Perkin Elmer Spectrum 100) at a resolution of 4 cm-1. Samples used for FTIR measurements were prepared by mixing the vacuum-dried samples with KBr (KBr: sample = 100: 1). The mixture was then ground extensively and pressed into a translucent disc. 2.5. Release behavior of Fe3O4/CaP/Alg with different ratios of P/Ca Fe3O4/CaP/Alg was synthesized with different ratios of P/Ca (0.6, 0.8, 1.6 and 2.4). Each sample with a different ratio of P/Ca was shaken in PBS with different pH values (pH = 5, 7.4, and 8.5) at 37 °C. The solution was centrifuged after a specific time lapse, and the supernatant was analyzed by PL (excitation: 470 nm, emission: 558 nm). The release percentage was calculated based on the following formula:

Release percentage % =

Dox in supernatant mg total amount of Dox in Fe O /CaP/Alg mg

2.6. Cell culture, viability testing, and observation A bladder cancer cell line (T24) was purchased from the National Health Research Institutes (NHRI), Taiwan. T24 cells were incubated in flasks with McCoy’s 5A at 37 °C, 5 % CO2, and 95 % humidified atmosphere and were sub-cultured every 3 days. Every 100 mL of McCoy’s 5A was supplemented with 10 mL FBS, 2 mL NaHCO3, 1 mL L-glutamine, and 1 mL Pen-Strep Sol. (penicillin 10000 units/mL and streptomycin

10

mg/mL).

Cell

viability

was

measured

by

a

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. T24 cells were seeded onto 96-well plates at a density of 2 × 104 cells well-1 and allowed to attach overnight. The medium was then removed, and each well was washed twice with 200 µL PBS. Media containing samples in various concentrations were added to each well, and the cells were incubated at 37 °C for 24 h. The medium was then 10 ACS Paragon Plus Environment

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removed, and the wells were washed twice with 0.2 mL PBS. 20 µL of MTT solution (5 mg/mL in DI water) was added to each well, and the cells were incubated for an additional 4 h. The medium was then replaced with 150 µL dimethyl sulfoxide (DMSO). The plates were left to stand for 4 h to dissolve the blue crystals, and the absorbance was recorded by a microplate reader at a wavelength of 570 nm. Cell viability was expressed as the average absorbance of the treated samples, relative to the untreated ones. 2.6. The effect of targeting and magnetic guidance on the drug delivery system T24 cells were seeded onto 4-well Lab-Tek slides at a density of 1 × 105 cells per well. After incubation at 37 °C with 5 % CO2 overnight, the medium was removed, and the slides were washed twice with PBS. To each well 0.25 mL DI water containing Fe3O4/CaP/Alg or RGD-Fe3O4/CaP/Alg samples (1 mg/mL) and 0.25 mL McCoy’s 5A were added. An external magnetic field induced by a magnet under the slides was used to guide the Fe3O4/CaP/Alg NPs. After incubation for an additional 4 h, the supernatant was removed, and the slides were washed extensively with PBS. 1 mL of neutral buffered formalin (10%) was then added to each. After 12 h, the neutral buffered formalin was replaced with Prussian blue staining reagent (20 % HCl(aq) and 10 % K4Fe(CN)6(aq)). After reaction for 20 min, the reagent was removed, and the slides were washed with DI water three times. After the removal of DI water, 500 µL of nuclear fast red counter stain was added and reacted for 5 min. Finally, the slides were thoroughly washed with DI water and placed under an optical microscope (Nikon eclipse-80i). Red and pink colors were produced by the nuclei and cytoplasm, respectively, and the blue color was emitted from Fe3O4. For another similar experiment, T24 cells cultured on slides were treated with Dox or RGD-Fe3O4/CaP/Alg under the same conditions as previously. The external 11 ACS Paragon Plus Environment


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magnetic field was utilized to guide the NPs. After staining with nuclear fast red, the cells were observed under an optical microscope. 2.6 Quantification of RGD-Fe3O4/CaP/Alg and Fe3O4/CaP/Alg inside cells The amount of iron element inside the cells was determined using inductively coupled plasma – mass spectroscopy (ICP-MS; Perkin-Elmer Elan-6000) after lysis of the cells. The cells were cultured as mentioned later. The cells were transplanted into 24 wells with 5 × 104 cells per well for one day. After washing several times with PBS, 0.5 mL of Fe3O4/CaP/Alg NPs or RGD-Fe3O4/CaP/Alg NPs containing solution (1 mg/mL) was added into each well, and the cells were cultured for another 4 h. 600

µL of HCl (12 M) was added into each well for both samples and the wells were stained for 2 h to dissolve the Fe3O4 nanoparticles. 600 µL of HCl (6 M) was used to wash the wells. A total of 6 mL of solution (5 wells for each sample) was collected into a vial for measurement. A single well in which the cells were cultured without nanoparticles was used to count the number of cells. The weight of Fe3O4 in each well, as measured by ICP-MS, was divided by number of cells to find the average weight of Fe3O4 NPs inside each cell.

3. Results and discussion 3.1 Synthesis and characterization of Fe3O4/CaP/Alg nanoparticles The concentration of alginate and the ratio of P/Ca in solution are two important key factors in the synthesis of Fe3O4/CaP/Alg nanoparticles during the pre-gel process. Our previous results have shown that the iron oxide nanoparticles could be synthesized with a suitable hydrodiameter of around 120 nm with good dispersity (i.e. polydispersity index, PDI = 0.25) after optimization of the synthetic conditions.29 In this study, various ratios of P/Ca were tested to form Fe3O4/CaP/Alg nanoparticles 12 ACS Paragon Plus Environment

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with a controllable particle size and surface charge. The values of the hydrodiameter and zeta potential of samples are shown in Fig. 1a, and the results clearly indicate that the hydrodiameter was increased and the zeta potential of samples became less negative with increasing concentration of calcium in the system. The decreasing magnitude of the zeta potential was attributed to the chelation of guluronic acid with calcium ions. The hydrodiameter increased greatly as the concentration of calcium exceeded 0.08 M, which suggests the particles aggregation. This can be evidenced by the increase in the PDI (as shown in Table S1 in the Supporting Information). It has been reported that the hydrodiameter of nanoparticles should be small enough to prevent aggregation in blood vessels or organs.31 Based on this principle, the concentration of calcium was chosen as 0.06 M, so as to achieve a hydrodiameter smaller than 200 nm and a narrow particle size distribution (i.e. PDI around 0.2). The effect of the P/Ca ratio was then investigated by fixing the concentration of calcium at 0.06 M. The hydrodiameter and zeta potential of the obtained Fe3O4/CaP/Alg nanoparticles with various ratio of P/Ca are shown in Fig. 1b. It seems that the ratio of P/Ca has little effect on either the hydrodiameter or the zeta potential. The semi-quantification of the elemental distribution by energy dispersive spectroscopy (EDS) (Table S2) demonstrates that the ratio of P/Ca in the samples increased, while the hydrodiameter of Fe3O4/CaP/Alg remained similar, as the ratio of P/Ca in the feed was increased. Here we fixed the ratio of P/Ca at 0.6 in order to obtain a smaller particle size.



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Figure 1. (a) The effect of the concentration of Ca2+ on the hydrodiameter and zeta potential of Fe3O4/CaP/Alg. The ratio of P/Ca was 0.6 for all cases. (b) The effect of the ratio of P/Ca on the hydrodiameter and zeta potential of Fe3O4/CaP/Alg as the concentration of Ca2+ was 0.06 M. To visualize the morphology, particle size, and crystalline structure of the synthesized Fe3O4/CaP/Alg samples, we conducted SEM and TEM. As shown in Fig. 2(a-1) and (b-1), the particle size and morphology of Fe3O4/CaP/Alg were similar to those for Fe3O4/Alg, indicating that the thickness of the CaP shell was thin. The core/shell structure of the Fe3O4/Alg nanoparticles was evidenced by TEM images. Fig. 2(a-2) clearly shows black dots (i.e. Fe3O4 nanoparticles) covered with a light gray layer (i.e. alginate layer). The formation of such a core/shell structure was 14 ACS Paragon Plus Environment

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discussed in our previous paper.29 Here, we further synthesized CaP in the Fe3O4/Alg sample by the co-precipitation method.32-33 As shown in Fig. 2(b-2)), the color of the shell became deeper, demonstrating the successful growth of CaP shells in the Fe3O4/Alg sample by the chelation of calcium ions by guluronic acid in the alginate polymer, followed by co-precipitation of CaP after the addition of phosphate ions. The elemental analysis (data not shown) demonstrated that both P and Ca elements were distributed homogeneously in both areas (i.e. gray and black dots), and their amounts were higher than for Fe and O on the black dots. The X-ray diffraction XRD patterns of Fe3O4/CaP/Alg with various ratios of P/Ca are shown in Fig. S1. The diffraction peaks of Fe3O4/CaP/Alg indicate that the sample possesses the cubic spinel structure of Fe3O4. There was no characteristic peak of CaP, however, indicating the amorphous phase of the CaP shell.

Figure 2. SEM images (left) and TEM images (right) of (a) Fe3O4/Alg and (b) Fe3O4/CaP/Alg. In order to promote the delivery efficacy of drug carriers, RGD-containing peptides (i.e. c(RGDfK)K3) were labeled on Fe3O4/CaP/Alg by EDC/NHS chemical 15 ACS Paragon Plus Environment


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binding between the amine groups of the peptide and the carboxyl groups of the alginate.18 The hydrodiameter and zeta potential of the RGD-functionalized Fe3O4/CaP/Alg (i.e. 200 nm and -22.9 mV, respectively) were close to those of unlabeled Fe3O4/CaP/Alg (i.e. 185 nm and -23.9 mV, respectively), indicating that our Fe3O4/CaP/Alg material was suitable for further post-functionalization. Unlabeled Fe3O4/CaP/Alg and RGD-Fe3O4/CaP/Alg were analyzed by Fourier transform infrared (FTIR) spectroscopy to investigate the chemical binding and interactions between the components. As shown in Fig. 3, the absorption of stretching vibrations between Fe-O at 565 cm-1 showed the existence of Fe3O4 nanoparticles. The absorption of symmetrical and asymmetrical stretching vibrations between COO- at 1415 cm-1 and 1618 cm-1 showed the characteristic absorption of alginate. The absorption of bending vibrations between O-P-O at 581 cm-1, and the symmetrical and asymmetrical stretching vibrations between P-O at 960 cm-1 and 1047 cm-1 showed the combination of phosphate and calcium. The characteristic absorption of calcium phosphate at 960 cm-1 would shift to lower wavenumber as the calcium phosphate formed in amorphous phase.34-35 The FTIR spectra suggested that each Fe3O4 nanoparticle was encapsulated inside an amorphous CaP shell which was covered by an alginate polymer. The absorption of P-O within Fe3O4/CaP/Alg shifted to lower wavenumber, indicating that the CaP shell is amorphous, which is in accordance with the XRD pattern (Fig. S1). The stretching vibrations at 1680 cm-1 and 1535 cm-1 are two characteristic absorptions of C=O and N-H, respectively, from the peptides. Moreover, the stretching vibration at 1433 cm-1 is due to the absorption of C-C from phenylalanine, which is one amino acid in the sequence of c(RGDfK)K3 (Fig. S2). The presence of the RGD peptide label on Fe3O4/CaP/Alg is confirmed by overlapping of the characteristic peaks of c(RGDfK)K3 and RGD-Fe3O4/CaP/Alg. 16 ACS Paragon Plus Environment

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The characteristic absorption of RGD peptide on RGD-Fe3O4/CaP/Alg is comparatively weak due to the relatively small amount of peptide labeled on Fe3O4/CaP/Alg.

Figure 3. FTIR spectra of Fe3O4, Fe3O4/Alg, Fe3O4/CaP/Alg, and pure alginate polymer. To realize the active guidance of the synthesized samples by external magnetic field, the saturation magnetizations of Fe3O4/Alg and its derivatives were measured with a superconducting quantum interference device. The value of saturation magnetization is an indicative value for magnetic materials. The external magnetic field could not be utilized to guide the materials if the value of the saturation magnetization were lower than 30 emu/mg.36 Another important indicative characteristic of magnetic materials for biomaterial application is superparamagnetism, meaning that the material would have magnetism when applying a magnetic field but have no magnetism when the magnetic field is removed. This indicative characteristic of magnetic materials means that the nanoparticles can gather together in the body when the external magnetic field is applied and can be separated from the body when the external magnetic field is removed.29 Fig. S3 indicates that both Fe3O4/Alg and 17 ACS Paragon Plus Environment


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Fe3O4/CaP/Alg are superparamagnetic (i.e. no hysteresis loop). The values of saturation magnetization shown in Table S3 demonstrate that the saturation magnetization of Fe3O4/CaP/Alg is lower than that of Fe3O4/Alg. The weight percentage of Fe3O4 within the Fe3O4/CaP/Alg decreases after the formation of the CaP shell; the CaP shell itself does not have any magnetic properties, however. In other words, the reason for the lower value of Fe3O4/CaP/Alg is the decrease in the weight percentage of Fe3O4. 3.2 Drug loading and release behavior of Fe3O4/CaP/Alg nanoparticles In order to study the drug delivery efficacy of the Fe3O4/CaP/Alg nanoparticles, the loading capacity and release behavior of drug molecules with Fe3O4/CaP/Alg nanoparticles as drug carriers with various ratios of P/Ca were quantified. Calcium phosphate has been widely used as a pH-responsive drug carrier with the controlled release property.33, 37-38 Here, doxorubicin (Dox) molecules were encapsulated into the CaP shell of the Fe3O4/CaP/Alg nanoparticles during the synthesis of the CaP shell on Fe3O4/Alg nanoparticles. Table S4 shows the loading capacity and efficiency of the Fe3O4/CaP/Alg nanoparticles, indicating that the ratios of P/Ca have little effect on the loading efficiency and amount. The loading efficiency was about 80%, and the capacity was about 27 µg/mg. The positively charged Dox has a high affinity to the negatively charged alginate occupying the space between core and shell. It is suggested that the interaction between the Dox and the alginate enables the Fe3O4/CaP/Alg nanoparticles to encapsulate Dox molecules. Generally, the solubility of calcium phosphate strongly depends on the composition of calcium and phosphate. The dissolution of CaP is based on Ksp value of CaP which is in relation with concentration of Ca2+, PO43- and OH-. We loaded Dox into the CaP shell of Fe3O4/CaP/Alg nanoparticles with various ratios of P/Ca and compared the release 18 ACS Paragon Plus Environment

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profiles of Dox from the Fe3O4/CaP/Alg nanoparticles at different pH values. As shown in Fig. 4, solutions with three different pH values (i.e. 5, 7.4, and 8.5) were chosen for testing drug release, because pH 5 represents the simulated surroundings of endosomes in cancer cells; pH 7.4 represents the simulated cell growth environment, and pH 8.5 represents the simulated urea in the bladder. The release rate of Fe3O4/CaP/Alg with the ratio of P/Ca between 0.6 to 1.2 was faster in pH 5 and pH 7.4 than that in pH 8.5. As the ratio of P/Ca increased to 2.4, however, the release rate slowed down in PBS with pH = 7.4. These results demonstrate that the drug release behavior in the Fe3O4/CaP/Alg nanoparticles is pH responsive. Chow discussed the relationship between pH value and calcium phosphate with different crystalline phase.39 His results indicated that the relationship between the calcium phosphate and the pH value is a U-shaped curve, regardless of the phase of the calcium phosphate. He revealed that the lowest solubility of calcium phosphate was around pH 8−9. His comment does correspond with the release profiles shown in Fig. 4. More Dox molecules were released into solution as the solution became more acidic due to the increased solubility of the CaP shell. The results in Fig. 4 indicate that the stability of the CaP shell has positive relationship with the ratio of P/Ca. Although pH value of urea is assumed to be 8, the actual pH value is different in each patient. The choice of carrier for intravesical therapy depends on the actual pH value in the patient’s urea.



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Figure 4. The release profile of Dox/Fe3O4/CaP/Alg at different pH values, with the ratio of P/Ca (a) 0.6, (b) 0.8, (c) 1.2, and (d) 2.4.

3.3 The targeting property and magnetic guidance of RGD-Fe3O4/CaP/Alg To evaluate the effects of passive targeting and magnetic guidance on cell uptake, T24 cells were treated with RGD-modified Fe3O4/CaP/Alg assisted by an external magnetic field. The course of intravesical therapy is 2 hours,21 so it would be better if drug carriers can be taken up by cells within 2 hours. RGD-based amino acids have been denoted as an αvβ3-targeting moiety. It has been reported that αvβ3 receptors are expressed on the membranes of T24 cancer cells.40 To accelerate the uptake of Fe3O4/CaP/Alg, cyclic RGD peptide (i.e. c(RGDfK)K3, denoted as RGD) was labeled on the alginate layer of the Fe3O4/CaP/Alg nanoparticles for targeting. To verify the effect of RGD peptide on the cellular uptake, the concentration of ferric ions represented in terms of the relative amount of the Fe3O4/CaP/Alg nanoparticles inside the T24 cells was measured by ICP-MS (Table 1). In addition to ICP-MS 20 ACS Paragon Plus Environment

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measurements,

the

cells

treated

with

unlabeled

Fe3O4/CaP/Alg

and

the

RGD-Fe3O4/CaP/Alg nanoparticles were also qualitatively observed with an optical microscope (Fig. 5). The results in Table 1 show that the concentration of ferric ions in the cells treated with RGD-Fe3O4/CaP/Alg is three times higher than that in the cells treated with unlabeled Fe3O4/CaP/Alg, evidencing the enhanced cellular uptake from specific targeting. The blue stain in Fig. 5 comes from the formation of Prussian blue, representing the location of the Fe3O4/CaP/Alg nanoparticles. There were more blue stains around cells treated with RGD-Fe3O4/CaP/Alg (Fig. 5c) than those treated with

Fe3O4/CaP/Alg

(Fig.

5b),

indicating

that more

RGD-Fe3O4/CaP/Alg

nanoparticles had been incorporated in the cells. In contrast to the small volume in the vascular system, the volume in the bladder is about 50 mL during intravesical therapy.3 The large 3D space in the bladder makes contact between the targeting-moiety labeled drug carriers and the cancer cells very difficult. Therefore, the external magnetic field was utilized to guide the magnetic nanoparticles to the precise position within short period. The effect of the external magnetic field on cell targeting is shown in Fig. 5d. The blue stains shown in Fig. 5d were very much greater than in Fig. 5c, revealing that the RGD-Fe3O4/CaP/Alg is guided to the surroundings of the cells by the external magnetic field, and then the particles interact with the cells by the combination of the RGD peptide with αvβ3 receptor. The results are proof that the combination of the passive targeting moiety and the external magnetic field could further enhance the effective uptake of magnetic RGD-Fe3O4/CaP/Alg nanoparticles.

Table 1. The concentration of ferric (II) ions in T24 cells treated with RGD labeled and unlabeled Fe3O4/CaP/Alg materials. 21 ACS Paragon Plus Environment


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Figure 5. Optical images of (a) T24 cells, (b) T24 cells with Fe3O4/CaP/Alg, (c) T24 cells with RGD-Fe3O4/CaP/Alg, and (d) T24 cells with RGD-Fe3O4/CaP/Alg and guidance from magnet.

3.4 Cytotoxicity and drug delivery efficacy of Fe3O4/CaP/Alg To evaluate the cellular viability of the synthesized Fe3O4/CaP/Alg, T24 cancer cells were treated with various amounts of Fe3O4/CaP/Alg carriers and analyzed by MTT methods. As shown in Fig. 6a, the cell viability of T24 did not greatly decrease as the amount of Fe3O4/CaP/Alg sample increased, i.e. cell viability


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was as high as 80% even in a sample with a very high concentration (1000 µg/mL), indicating that our Fe3O4/CaP/Alg is biocompatible. In contrast to the high cell viability of T24 treated with the doxorubicin drug (Fig. 6b), the cell viability of T24 treated with Dox-loaded, RGD-functionalized, magnetically

guided

Fe3O4/CaP/Alg

nanoparticles

(denoted

as

RGD-Fe3O4/CaP(+)/Alg) showed that more than 60% of the cells died with the same amount of Dox (i.e. 5 µg/mL). The enhanced cell-killing ability of the RGD-Fe3O4/CaP/Alg could be attributed to the efficient active guidance by the magnetic field and passive guidance by RGD peptides.



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Figure 6. (a) Biocompatibility of Fe3O4/CaP/Alg and RGD-Fe3O4/CaP/Alg with T24 cells. (b) Cell viability of T24 cells treated with free DOX and Dox-loaded RGD-Fe3O4/CaP/Alg. Neutral red was used to stain living cells, and the stained cells were observed by optical microscopy. As shown in Fig. 7, most of cells treated with doxorubicin were still alive (Fig. 7a), which corresponds to the results shown in Fig. 6b. Cells treated with Dox-loaded, RGD-functionalized Fe3O4/CaP/Alg nanoparticles without and with a magnetic field were observed, and the results are shown in Fig. 7b and 7c, respectively. In contrast to the healthy cells in the area without a magnetic field (Fig. 7b), it can be clearly seen that most of cancer cells are dead in the area with the magnetic field (Fig. 7c). The results shown in Fig. 7c could be attributed to magnetic guidance of the Dox-loaded, RGD-functionalized Fe3O4/CaP/Alg nanoparticles which could accumulate most of particles within the magnetic region. The results also revealed the advantage of magnetic guidance, that magnetic drug carriers could be guided to the desired region and only release drug molecules in that area, indicating that the adverse effect of chemotherapy can be reduced effectively.


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Figure 7. Optical images of cells under the conditions of (a) free Dox only, (b) Dox-loaded RGD-Fe3O4/CaP/Alg outside the zone of external magnetic field, and (c) Do- loaded RGD-Fe3O4/CaP/Alg inside the zone of external magnetic field.

4. Conclusion In summary, this study reports the synthesis of core/shell/corona Fe3O4/CaP/Alg nanoparticles with a nanometer particle size and COOH functional surface through the combination of co-precipitation and pre-gel methods after optimizing the synthetic conditions. The synthesized Fe3O4/CaP/Alg exhibits excellent biocompatibility and a controlled release property. The abundant COOH groups on the external surface affords the ability to functionalize Fe3O4/Cap/Alg with an αvβ3-receptor targeting ligand (i.e., c(RGDfK)K3), leading to a specific cell-targeting ability. In addition, a chemotherapeutic drug (Dox) could be successfully loaded on/released from the RGD-functionalized Fe3O4/CaP/Alg. After applying an external magnetic field, the Dox-loaded, RGD-functionalized Fe3O4/CaP/Alg nanoparticles could be effectively accumulated in the target region, resulting in enhanced drug delivery efficacy. The Fe3O4/CaP/Alg-based nanoparticles can lead to a new generation of nanovehicles for drug delivery systems.

Supporting Information

Supporting Information contains summary of effect of Ca2+ and P/Ca to zeta potential and particle size of Fe3O4/CaP/Alg, XRD pattern and FTIR spectrum of Fe3O4/CaP/Alg, loading amount of DOX in carrier, and quantification of magnetization of Fe3O4/CaP/Alg.

Acknowledgments 25 ACS Paragon Plus Environment


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This research was supported by the Ministry of Science and Technology (MOST), Taiwan (103-2113-M-008-001; 104-2119-M-008-010), National Taiwan University (104R7706),

and

the

National

Health

Research

Institutes

of

Taiwan

(03A1-BNMP14-014). This work was partially supported by the Australian Institute for Innovative Materials (AIIM) Gold/2017 grant. Y.Y and Z.A.A. are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs. The authors would like to thank Dr Macs Bio-Pharma Private Limited for helpful contribution to biomedical applications.


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