An Integrative Folate-Based Metal Complex Nanotube as a Potent

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An integrative folate-based metal complex nanotube as a potent antitumor nanomedicine as well as an efficient tumor-targeted drug carrier Lixiang Liu, Bingxia Li, Qiyan Wang, Zhipeng Dong, Hongmei Li, Qiaomei Jin, Hao Hong, Jian Zhang, and Yue Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00520 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 2, 2016

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Bioconjugate Chemistry

An integrative folate-based metal complex nanotube as a potent antitumor nanomedicine as well as an efficient tumor-targeted drug carrier Li X. Liu†, Bing X. Li†, Qi Y. Wang†, Zhi P. Dong†, Hong M. Li†, Qiao M. Jin‡, Hao Hong§, Jian Zhang*‡, Yue Wang*† †

Key Laboratory of Biomedical Functional Materials, School of Sciences, China Pharmaceutical University, Nanjing 211198, Jiangsu Province, China. ‡

Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, Jiangsu Province, China.

§

Center for Molecular Imaging, Department of Radiology, University of Michigan-Ann Arbor, MI 48109-2200, United States. *Corresponding authors’ email: [email protected], [email protected]

ABSTRACT Metal-organic complexes (MOCs) are emerging developing functional materials, the different categories of metal ions and organic biomolecules provide great possibilities for the morphologies, sizes and properties of the products. Enlightened by the previous works of folatenickel nanotubes (FA-Ni NTs), herein, a series of metal ions are tested to coordinate with folate (FA) by solvothermal method, among which folate-cobalt (ΙΙ) complex is formed to be a scaffold

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for the nanotube with the length of 150-500 nm and inner diameter of 6-11 nm, while the other metal ions fail. In vitro experiments reveal that folate-cobalt nanotubes (FA-Co NTs) have excellent antitumor activity towards tumor cells with high expression level of folate receptor (FR), whereas show extremely low toxicity to normal cells. Furthermore, this kind of NTs do perform better antitumor ability when encapsulate the anticancer drug doxorubicin through cell surface receptor-mediated endocytosis. Moreover, we study the fundamental pharmacokinetic profiles and biodistribution of FA-Co NTs on mice and also prove its targeting capability to tumor tissues on tumor-bearing mice using radioactive iodine-131(131I) tracing method. FA-Co NTs can also markedly inhibite the growth of tumor with minimal side effects when administrated individually in vivo. These findings will expand the research on the FA based metal complexes nanomaterials as a kind of potential antitumor nanomedicine as well as a targeted drug carrier.

INTRODUCTION Over the last decade, metal-organic complexes (MOCs) have drawn much attention because lots of organic molecules can coordinate with various metal ions to form metal complexes with diverse geometries, providing infinite possibilities of coordination style and wide applications. In the construction of MOCs, organic molecule ligands always play a decisive role in forming the title materials. The different linking sites of ligands would tune the linking model and directionality of the complexes. With the development of nanotechnology and nanomaterials, MOCs have been further explored and put forward to assembly the related nano-sized materials bearing more functional properties. Lee and co-workers1 reported construction and structural modification of different morphologies of one-dimensional coordination polymers focused on the bulk crystalline solids. Xing and co-


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workers2 designed a simple pH-responsive release system by integrating a metal ion and ligand or self-assembling these species with biodegradable host molecules to form nanoparticles with metal-ligand or host-metal-ligand architectures. Liu and co-workers3 reported rational design of nanoscale metal-organic frameworks (NMOFs) composed by hafnium (Hf4+) and tetrakis (4carboxyphenyl) porphyrin (TCPP) to achieve a remarkable antitumor effect of photodynamic therapy (PDT) and radiotherapy (RT). These evolutionary MOCs materials are promising and may enrich the applications of MOCs in much more areas, such as catalysis, disease diagnosis and cancer treatment. However, to the best knowledge of us, some objective problems promoted for them cannot be ignored: (1) The synthesis condition of these materials is relatively harsh; (2) The linkers used are limited in polypyridyl molecules; (3) The shape and size control of products is terrible; (4) The post treatment of the materials is tedious; (5) Some undesired bio-toxicity is inevitable. Given these, our group recently have focused on the biomolecule-based coordination-polymer assemblies to achieve the functional material in nanoscale4. As a kind of natural ligand, biomolecules have received great favors gradually for their inherent biological properties, such as high affinity and strength, biodegradability and no toxicity, hence, they are considered to be an ideal candidate defining the possible MOCs with excellent performance. Among them, folate (pteroyl-L-glutamic acid, denoted as FA), a representative small organic biomolecule, is highly appreciated for its unique highlights. As a member of vitamin B, FA has good properties including low molecular weight, biocompatibility, no immunogenicity, low cost and high affinity with folate receptor (FR), which is a highly selective tumor marker overexpressed in greater than 90 % of ovarian carcinomas. In addition to these advantages, FA molecule has many active sites to be a typical ligand to coordinate with some metals5. As reported by Sudimack et al.6, the


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carboxyl groups (-COOH) of α and γ sites in FA are relatively active and readily to coordinate with metal ions. Hamed and co-workers7 reported that FA could coordinate with some transition metals such as Fe (ΙΙΙ) and Cu (ΙΙ) through -COOH under special conditions, but the morphologies and sizes of these coordination complexes were irregular and erratic, which had a great relationship with the coordination condition. Our previous works have reported a kind of novel folate-nickle biomolecule-based coordination nanotubes with perfect antitumor activity4. In contrast to the popular folate-drug conjugates8, the synthetic method of this complex nanotube is simple and convenient to make the regular shape and uniform morphology. Moreover, we need not to do any tedious post treatment to achieve the efficient targeting and biocompatible of folate-nickle nanotubes (denoted as FA-Ni NTs). Inspired by this, we are much interested in studying the metal complexes in nanoscale using FA as a ligand to further investigate the morphologies and composition of these nanomaterials, which might be helpful to expand the type of metal nanomedicine. All of these products were characterized by transmission electron microscope (TEM), X-ray powder diffraction (XRD), fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). As expected, FA was found to coordinate with Co2+ to form tubular structure in a similar way with Ni2+, while the other metal ions are not inclined to participate in the coordination. In terms of its tubular structure, we focused on the investigation and evaluation of antitumor activity in vitro of the folate-cobalt complex nanotubes (denoted as FA-Co NTs, see Scheme 1). We also used radioactive iodine-131 (131I) tracing method to determine the in vivo fundamental pharmacological profiles of FA-Co NTs on mice and tumor targeting capability on tumorbearing mice. Furthmore, the preliminary inhibition function of tumor growth of FA-Co NTs was investigated when administrated individually.


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Scheme 1. Schematic illustration of FA-Co NTs as a nanomedicine and a drug carrier. RESULTS AND DISCUSSION Synthesis and characterization The general preparation strategy for these FA-involved materials is identical, but only FA-Co complex was found to form the apparent tubular structure. The shape and size of all the products were characterized by TEM (Fig 1). TEM images in Fig 1a clearly showed the nanotubular morphology in case of CoCl2 mixed with FA (10-20 nm for outer diameter, 6-11 nm for inner diameter, 4-9 nm for wall thickness and 150-500 nm for the length). Fig 1c, 1e and 1f (corresponding to the product of FA mixed with Zn2+, Fe2+ and Cu2+) showed the spherical nanoparticles with an average size of 21-50 nm, 5-40 nm, 10-30 nm respectively, while Fig 1d (product of FA mixed with Ca2+) showed the amorphous and irregular aggregation cluster. To improve the dispersibility and reduce the length of FA-Co NTs for the further application, we chose ultrasonic cell crusher (JY98-ΙΙΙ) to post treat the FA-Co NTs. TEM confirmed the primary tubular structure and reduced length (50-250 nm) of FA-Co NTs, while the other parameters were unchanged (Fig 1b), which were used for the following experiments.


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Fig 1. TEM images of (a) original FA-Co NTs, (b) treated FA-Co NTs, (c) ZnCl2 reacted with FA and 10 mL 85 % N2H4•H2O, (d) CaCl2 mixed with FA and 10 mL 85 % N2H4•H2O, (e) FeCl2 mixed with FA and 5 mL 85 % N2H4•H2O, (f) CuCl2 mixed with FA and 5 mL 85 % N2H4•H2O. The chemical composition of the products was determined by XRD, and Jade 5 was used for qualitative analysis. Fig 2 (blank line) had either no typical diffraction peaks of cobalt compounds or FA molecule, which just displayed the non-crystalline property of FA-Co NTs. Comparing with the standard pattern (JCPDS), we can easily differentiate the other species of the nanoparticles (Fig 2). It means that FA is able to coordinate with Co (ΙΙ), which constructs the scaffold of the nanotubular structure and has high similarity with reported FA-Ni NTs. This result is coinciding with the FTIR data (see supporting information in Fig S1) that the C=O absorption band at 1694 cm−1 of free FA disappears, while νas (COO-) and νs (COO-) absorption bands at 1570 and 1453 cm−1 offset respectively, indicating that FA has coordinated with Co2+.


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In addition, the small peak of the spectra at 968 cm−1 is typical for the N-N stretching of the hydrazine ligand18 and further proves cobalt-hydrazine coordination. Elemental analysis and inductively coupled plasma emission spectrometer, furthermore, determined the content of C, H, N and Co elements in FA-Co NTs, they were C: 32.84 wt%, H: 4.85 wt%, N: 24.49 wt% and Co: 16.20 wt%, which was similar with the reported work of FA-Ni NTs. However, the other metals are not inclined to form the complexes with FA, just producing the corresponding metal oxide at the solvothermal reaction condition.

Fig 2. XRD pattern of FA-Co NTs, ZnO nanoparticles, CaO nanoparticles, Fe3O4 nanoparticles and CuO nanoparticles.


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Fig 3. XPS spectra of FA-Co NTs: (a) full scan, (b) C1s, (c) N1s, (d) O1s and (e) Co2p.


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For Ni and Co are both the VIII group elements, as well as the structure similarity of the FA-Ni NTs and FA-Co NTs, we deduced that the main coordination mode of this FA-Co NTs should be same with the reported FA-Ni NTs. XPS studies were performed to further demonstrate the energy position expected for FA-Co NTs. Co 2p peak in the XPS survey spectra confirmed that all the Co element in the sample were Co (ΙΙ) with a characteristic core level peak of Co 2p1/2 and Co 2p3/2 near 796 eV and 780 eV, respectively19 (Fig 3e). Fig 3b-3d showed C1s, N1s and O1s spectra. The most noteworthy was that in Fig 3c, the N1s band of a single peak at 399 eV attributed to N-Co, identical with other known Co (ΙΙ) complex with hydrazine (∼399.4 eV)20. Based on these results, the N1s peak at 399 eV in the XPS of the FA-Co NTs may attribute to the coordination of N2H4•H2O with Co (ΙΙ), which fits with FTIR results and evidences the involvement of N2H4•H2O. In this study, we tried to make FA molecule coordinate with various metal ions to form nanosized complexes using solvothermal method. Due to the unequal coordination abilities of each metal ion with FA molecule, Ca2+, Zn2+, Fe2+ and Cu2+ just formed the corresponding oxides instead of the complexes. As known to us, Co2+ as well as Ni2+, which belong to VIII group element, tends to form inner-orbital coordination compounds, while the others are out-orbital coordination compounds, which are related to the outer electron state. The former has distinct advantages over the later on strength of covalent bonds and stability. It is worth mentioning that in the reaction process of FA-Co NTs, the system was preheated at 120 °C for 2 h before adding N2H4•H2O as a co-ligand, otherwise it was hard to form the tubular structure. This fact supports the key role of the N2H4 group in the formation of NTs. It has been clearly illustrated in our previous work on FA-Ni NTs. The drug release behavior of FA-Co NTs in vitro


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The unique nanotubular structure with the negative zeta potential (-15.4 mV, see supporting information in Fig S2) is considered to be a good candidate for the drug delivery22. The entrapment of drugs into the FA-Co NTs was confirmed by absorption measurements. The results showed that DOX had a pretty high drug loading efficiency of approximately 98.1 % and the drug loading content was nearly 38.01 % (DOX standard curve: y=17.11x-0.0408, y: absorbance value at 490 nm, x: concentration of DOX, from 0.001-1 mg/mL). The hollow tubular morphology really allows a high drug storage capacity and the FA-Co-DOX NTs performs a similar negative zeta potential (-14.6 mV, see supporting information in Fig S2) with bare FA-Co NTs. Details of the calculation method are provided in the Experimental Section. The release of the drug has been studied by using dialysis method through incubating FA-CoDOX NTs in pH 5.5 and 7.4 solution at 37 °C, which mimics the lysosomal compartment in cancer cells and the normal human blood respectively23. Results showed that FA-Co-DOX NTs released DOX about 48.0 ± 4.05 % in 72 h at pH 5.5 (Fig 4), while only about 13.6 ± 2.03 % in 120 h at pH 7.4. A noticeable phenomenon is that the release rate at pH 7.4 is significantly lower than at pH 5.5 in the first 24 h, which proves the protective effect of NTs for the drug and is quite useful for the receptor-mediated endocytosis and thus reducing toxicity to the normal tissues since single DOX diffuses into external matrix freely and reaches peak value during extremely short time24, its manner is greatly different from DOX loaded NTs. The local sudden release of DOX from FA-Co NTs at pH 5.5 was due to the small part of the adsorbed DOX on the surface of FA-Co NTs, which was then released quickly into the buffer solution via free diffusion within several hours caused by increased protonation of NH2 groups on DOX. These pH-responsive release behavior is inspiring to show higher DOX release from FA-Co-DOX NTs in a slow and sustained manner over 72 h at pH 5.5 compared to physiological pH 7.4. Herein we


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would venture to guess the release behavior of DOX, that is at the first stage, the coordination bonds in FA-Co NTs are sensitive to acidity, especially -N2H4 group involved in the coordination bonds is more likely to be etched and broken, leading to the decomposition of the FA-Co NTs, although the process is relatively sluggish. At the second stage, DOX diffuses into the cytoplasm and cell nucleus from FA-Co NTs due to its solubility at different pH25,31.

Fig 4. In vitro release profiles of DOX from FA-Co-DOX NTs at pH 5.5 and pH 7.4. After drug release, we re-collected the residue left in the dialysis membrane to check the morphology of FA-Co NTs. The images (see supporting information in Fig S3) showed that certain NTs indeed were destroyed after the acid corrosion for 24 h. In contrast to the original FA-Co NTs (Fig 1a and 1b), the open ends of the NTs disappeared and finally turned into smaller pieces (Fig S3b), which might be helpful for the drug release. For pH 7.4 group, the NTs could keep tubular structure and size even for 120 h, showing good stability of FA-Co NTs under physiological acid-base condition. In vitro cytotoxicity research Inspired by the excellent antitumor activity of its analogue FA-Ni NTs, herein we expected to assess the antitumor potency of FA-Co NTs as well as FA-Co-DOX NTs. HeLa, A549 and L-O2


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cells were treated with FA-Co NTs and FA-Co-DOX NTs for 72 h (DOX, CoCl2 or FA were used as the control groups). MTT assay results showed that FA-Co NTs displayed good antitumor activity (IC50 of HeLa is 1.47 µg/mL, IC50 of A549 is 5.43 µg/mL, IC50 of L-O2 is 20.32 µg/mL, Fig 5), which is very near the DOX against HeLa (IC50 of HeLa is 1.05 µg/mL), but its cytotoxicity on normal cells is much lower than the DOX (IC50 of L-O2 is 4.51 µg/mL). As control, FA, CoCl2 and their mixture in the solution showed no inhibition activity. The extra efficacy of the antitumor activity of FA-Co NTs in vitro may attribute to its specific nano-sized tubular morphology because some of the NTs could successfully escape from lysosome duo to the sponge effect of -NH2 group in FA molecule. However, the mixture of CoCl2 and FA (at the same ratio with NTs) in the solution state has no inhibition at all although the complex formation could be possible in the solution, the antitumor mechanism of small molecule and nanomaterials should be completely different thus resulting in the distinctive inhibition result26，but powerful evidence needed to be explored.

Fig 5. Cell inhibition rate of HeLa cell lines (black), A549 cell lines (red) and L-O2 cell lines (blue) after co-incubation with FA-Co NTs for 72 h respectively. As expected, FA-Co-DOX NTs (DLC = 38.01 %) also effectively inhibited the tumor cell growth as expected (IC50 of HeLa is 1.21 µg/mL, IC50 of L-O2 is 16.76 µg/mL). All these data


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indicate that FA-Co NTs can specifically target toward the HeLa cells with FR mediated endocytosis, then it slowly released loaded DOX, just like we stated in the section of in vitro drug release behavior of FA-Co NTs, and multi-strategy to kill tumor cells. In case of the L-O2 cells with a lower expression of FR, NTs vehicle showed much lower cytotoxicity than free DOX because of the low target delivery and the controlled release. Confocal laser scanning microscopy (CLSM) imaging and flow cytometry analysis In this section, we used fluorescein FITC to label FA-Co-DOX NTs (FA-Co-DOX-FITC NTs) to realize co-localization of FA-Co NTs. Fig 6 showed the cell uptake and intracellular distribution of free FITC, DOX and FA-Co-DOX-FITC NTs in HeLa cells after incubation for 4 h. The free FITC was rarely distributed inside the cell because such a small molecule could not easily permeate the cell membranes (Fig 6a). Free DOX was primarily accumulated in the nuclei of the HeLa cells where it could lead to cell death by interrupting DNA replication21,27 (Fig 6b). FACo-DOX NTs were mainly localized in cytosol and dot-shaped fluorescence was observed within cytoplasm. DOX then was localized in both cytosol and nuclei of the HeLa cells because it was delivered by NTs and taken up by the cells through a non-specific endocytosis mechanism and the DOX released from FA-Co NTs could be transported into cells and accumulated in the nuclei via diffusion (Fig 6c), while in Fig 6d for L-O2 cells, it was relatively hard for FA-Co-DOX NTs to pass through the cytomembrane and enter into the nuclei, so less fluorescence could be observed.


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Fig 6. CLSM images: (a) and (b) were single FITC (collected at 530 nm) and DOX (collected at 488 nm) treating with HeLa cells for 4 h (the blue nucleus stained by DAPI collected at 360 nm;


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right: merged images), (c) and (d) were 10 µg/mL of FA-Co-DOX-FITC NTs treating with HeLa and L-O2 cells for 4 h respectively. To quantify the positive/negative ratio based on the average FITC fluorescence signal/intensity to determine the specificity/selectivity of the FA-based targeting system, flow cytometric analyses of FA-Co-FITC NTs in HeLa and L-O2 cell lines were carried out. Results showed that the FA-Co-FITC NTs were carried into the HeLa cell lines at an uptake of about 98.3 % and the L-O2 cells lines at about 40.6 % (Fig 7) for comparison via FA binding to FR and other endocytosis mechanism respectively28. Taken together, both results clearly demonstrated that FA-Co NTs had efficient targeting on HeLa cell lines with high specificity mediated by FR and minimal nonspecific binding on L-O2 cell lines.

Fig 7. Flow cytometric analysis of FA-Co-FITC NTs in HeLa (left) and L-O2 (right) cell lines for 4 h. ,

Our results highly coincided with Chen s work29 about molecular recognition of FA by FR, in which he expounded that FR (especially FRα) had a globular structure stabilized by eight disulphide bonds and contained a deep open folate-binding pocket comprised of residues that


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were conserved in all receptor subtypes, while the pteroate moiety in FA was buried inside the receptor, its glutamate moiety was solvent-exposed and sticked out of the pocket entrance, allowing it to be modified simultaneously without adversely affecting FR binding. As proved above, FA could coordinate with Co2+ just making use of its -COOH, which provided great convenience for the post modification of FA-Co NTs but not affected its binding ability with FR. Radiolabeling and in vitro stability The Iodogen method used for radioiodination of FA-Co NTs was simple and effective in the experiment. Paper chromatography analysis showed that labeling rate of FA-Co NTs was greater than 95 % at 45 °C for 2 h. In addition, the radiochemical purity of 131I-FA-Co NTs assayed after 24 h incubation with serum at 37 °C was almost invariant (about 87 %), which was suggestive of the excellent stability in physiological conditions (Fig 8a) and therefore, the radioactive counts of 131

I would be able to reflect the in vivo biodistribution and pharmacokinetic behavior of FA-Co

NTs. Pharmacokinetic study The mean plasma radioactive concentration-time profile of 131I-FA-Co NTs was shown in Fig 8b and the major pharmacokinetic parameters determined by the two-compartmental model30 (c = Ae-αt + Be-βt, c is the concentration of mean plasma radioactivity) was presented in Table 1 after data fitting and calculation. Table 1. Main pharmacokinetic parameters of FA-Co NTs on mice. Parameter

Units

Value for rats

AUC(0-t) T1/2α T1/2β Cmax

MBq/L*h h h MBq/L

353.25 0.53 16.04 61.25


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CL Vss MRT

L/h/kg mL h

0.06 1.18 20.84

The first-phase half-life (T1/2α) of FA-Co NTs is about 0.73 h, indicative of that FA-Co NTs reached the distribution balance between blood and tissues quickly. The calculation results show that clearance (CL) is 0.06 L/h/kg for intravenous injection, which is relatively moderate for a drug. Furthermore, the eliminative half-lives (T1/2β) of the injection mode is 16.04 h, it is advantageous in the view of safety and rather long among various drug delivery carriers31. The value of steady-state volume of distribution (Vss, 1.18 mL) is similar to the total body water content in mice (0.7 mL/g), illuminating the secondary distribution behavior of FA-Co NTs in blood and extracellular fluid. The results of Fig 8b indicated that NTs was eliminated almost completely from the blood after 24 h, which was consistent with the constantly low radioactivity of kidney and bladder (Fig 9a).

Fig 8. (a) The radiolabeling stability of

131

I-FA-Co NTs in mouse plasma at 37 °C for 24 h, (b)

The mean plasma radioactive concentration-time profiles following a single intravenous administration of 14.8 MBq/kg of 131I-FA-Co NTs. Biodistribution study


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The biodistribution results on mice were shown in Fig 9a with the data expressed as mean % ID/g ± SD in view of the variable amount by intravenous injection. All the radioactivity of

131

I

was present in measurable levels throughout the entire body. From Fig 9a (Detailed values were in supporting information Table S1) we could clearly conclude that there was negligible accumulation of 131I-FA-Co NTs in the brain for the reason that it could not effectively pass the blood-brain barrier. At 30 min p.i, we found that lung and liver had high radioactivity compared with other tissues and themselves in different time points, it was in agreement with the value of T1/2α and owing to the retention function of reticuloendothelial systems (RES) responsible for the clearance of exogenetic nanomaterials upon systemic administration32. Taking lung as an example, a portion of FA-Co NTs would be captured by the pulmonary capillary bed due to their size and then accumulated in the lung, while some parts of them would pass through the alveolar wall of the capillary barrier and entered into the blood circulation and other tissues33. Relatively high uptake of spleen, muscle and skin is positively correlated with the content of

131

I-FA-Co

NTs in the blood at different time points, showing that FA-Co NTs moved freely with the blood and did not accumulate longer in these tissues. In the other organs, such as the stomach, kidney and intestine, the uptake of

131

I-FA-Co NTs was close to being saturated during 30 min after

post-dosing. All these results support the fact of rapid distribution of FA-Co NTs among blood and tissues during short time after p.i. However, As time going on, the accumulation of most organs and tissues reached peak value (1 h in Fig 9a) and then put up a well-bedded clearance of radioactivity within 24 h except lung and liver at 4 h, which performed another peak value and may be on account of the combined effects of RES function and/or the possible degradation of FA-Co NTs and/or partial detachment of 131I labeling from FA-Co NTs in vivo34. The high levels of

131

I found in the kidney after 30 min and the rapid decline in the overall radioactivity levels


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thereafter indicated that most of the NTs were eliminated through the renal excretion route and into urine. In vivo tumor targeting and tumor inhibition of FA-Co NTs Encouraged by in vitro excellent antitumor activity and pharmacological profiles on mice of FACo NTs, we then chose HeLa cells to establish subcutaneously implanted tumor model on nude mice. When the tumor volume reached about 100-200 mm3 (see the growth curve of tumors in supporting information Fig S4), mice were used for the following experiments. As prepared

131

I-FA-Co NTs were intravenously injected into HeLa tumor-bearing mice and for

quantification analysis of tumor uptake of NTs, we collected major tissues of mice at 1 h, 4 h and 24 h for radiocounting, results were showed in Fig 9b (Detailed values were in supporting information Table S2). At 1 h p.i, we found the tumor accumulation was 1.91 ± 0.53 % ID/g, it was relatively high, while other tissues had similar distribution with on normal mice. As time increased, the % ID/g in other tissues declined obviously because of the gradual clearance of FACo NTs in vivo, while kept long retention in tumor sites, clearly indicating that FA-Co NTs could accumulate in tumor tissues by passive targeting of EPR effect as to solid tumor35, then increased tumor uptake through active targeting of FR on the surface of tumor cells. On this base, we then investigated the preliminary tumor inhibition effect of FA-Co NTs. Tumor models on nude mice were established using the method in Experimental Section. We periodically measured the tumor sizes after tail injection of DOX and FA-Co NTs. The mice treated with saline were used as the control group. We measured the tumor sizes at different time points in order to investigate the tumor growth after administration. Fig 9c showed that in vivo the growth of tumor was markedly inhibited when treated with DOX and FA-Co NTs. Moreover, NTs group performed a better curative effects than pure DOX. After treatment with DOX and


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FA-Co NTs for 16 days, we collected all the tumor tissues in each group and weighed them, getting the similar tumor inhibition for DOX and FA-Co NTs, respectively. To our knowledge, DOX, a classical chemotherapeutic agent, has low tumor selectivity and could not interact with tumor cells in vivo for the reason that it only retains a transient high plasma drug concentration for a short time and is quickly excreted out from body after injection36, but to FA-Co NTs, it performed a distinct accumulation in tumor tissues owing to EPR effect when NTs were injected into the mouse blood, then FR mediated the NTs entered into cells and NTs played a role. We also measured the changes of mice weights during the treatment process, and the initial weights of nude mice were recorded to be approximately 20.1 ± 0.21g. As shown in Fig 9d, the mice weights of all the three groups increased in different degree, they were 23.7, 21.3 and 22.9 g on average for saline, DOX and FA-Co NTs group at the end of treatment, respectively. The results indicated that FA-Co NTs had a good biocompatibility and no obvious toxicity for mice.


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Fig 9. (a) Biodistribution histograms of 131I-FA-Co NTs on mice, (b) Biodistribution line chart of 131

I-FA-Co NTs on tumor-bearing mice, (c) Real-time observation of tumor sizes in vivo after

treatment with samples. Error bars represent means ± SD, (d) Real-time weight analysis of mice after each treatment. Error bars represent means ± SD. CONCLUSION In this study, we utilize FA to react with different metal ions via solvothermal procedure, and results show that only Co2+ can coordinate with FA to fabricate a new-type metal complex nanotube, which expands the type of metal-FA coordination nanotube. In vitro experiments indicate that FA-Co NTs can target tumor cells and have excellent antitumor activity with lower toxicity to normal cells than DOX. Furthermore, the remarkable hollow tubular structure of FA-


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Co NTs has the excellent capacity to make it be an ideal drug carrier (Drug loading content of DOX is 98.1 %, drug loading efficiency is 38.01 %). FA-Co NTs are pH-responsive because when they target tumor sites, the coordination bonds are readily broken so that the tubular structure is destroyed at the acidic tumor microenvironment, thus releasing loaded drugs to achieve the more efficient antitumor activity. In vivo experiments have proved that FA-Co NTs have excellent tumor targeting and tumor inhibition function on tumor-bearing mice. Encouraged by the promising results, in the following studies, we are going to further investigate biological behaviors of the FA-M NTs and FA-M-drug delivery systems (M = Ni and Co) in vivo, which might enable the development of them in the future clinical application. EXPERIMENTAL SECTION Chemicals and apparatus All reagents and solvents were commercially available and used without additional treatment. Folate (97 %) were purchased from Jiaxing Biochemicals Corporation, CoCl2•6H2O, anhydrous ZnCl2, CaCl2•2H2O, FeCl2•2H2O, CuCl2, N2H4•H2O (85 %, analytically pure) were obtained from Shanghai National Medicine Corporation, anhydrous ethanol, Doxorubicin•HCl (DOX•HCl, denoted as DOX, 99.8 %) and fluorescein isothiocyanate (FITC) were purchased from Melone Pharmaceutical Corporation. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin-EDTA, penicillin-streptomycin, dimethylsulfoxide (DMSO), 4,6-diamidino-2-phenylindole (DAPI) were obtained from Gibco. 96 well plates, 6 well plates and 10 mL graduated sterile centrifuge tubes were purchased from KeyGen BioTECH. Sodium iodide (Na131I) were supplied by HTA Co (Ltd, Beijing, China) and have the radionuclidic purity over 99 %. Other reagents and chemicals were at least analytical reagent grade.


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Transmission electron microscope (TEM) were performed on a JEOL 2100 with accelerating voltage of 200 kV. TEM samples were prepared by drop-casting dispersion in ethanol solutions onto copper grids covered by carbon film and dried at ambient temperature. The non-crystalline structure of the prepared products was identified by X-ray powder diffraction (XRD) with a Bruker D8 diffracto-meter system using a Cu Kα radiation source (λ = 0.15406 nm) as the X-ray source in the 2θ range of 10-90o. Jade 5 was used for qualitative analysis. Fourier transform infrared spectroscopy (FTIR) (4000-400 cm−1) were recorded on Bruker FTIR using KBr pellets, the resolution is 2.0 cm−1. X-ray photoelectron spectroscopy (XPS) analysis was performed to determine the elemental composition and oxidation state of materials using Al Kα X-ray at 15 kV and 15 mA. The standard C1s peak (Binding energy, Eb = 284.80 eV) was used to eliminate the static charge effects. The surface charge of the nanocarrier was investigated on Malvern Zetasizer Nano ZS 90 zeta potential analyzer. Ultraviolet-visible (UV-vis) spectra were collected using a LAMBDA-35 spectrometer. Confocal images were acquired using a Zeiss confocal laser scanning unit mounted on an LSM 710 fixed-stage upright microscope (CLSM). Flow cytometry experiments were performed with a BD FACS Aria ΙΙ apparatus. Radioactive counts were measured in an automatic gamma counter (SN-695; Hesuo Rihuan photoelectric instrument, Shanghai, China). Preparation of FA-Co NTs In a typical experiment, FA (0.5 mmol) and CoCl2•6H2O (1 mmol) were added to a mixture of ethanol and H2O with stirring and ultrasonicated for 10 min, the mixture immediately was transferred to a poly-tetrafluoroethylene-lined autoclave with heating at 120 °C for 2 h, and then cooled naturally to room temperature, after that N2H4•H2O (20 mL, 85 %) was added to the mixture with continuous stirring and heated at 120 °C for 12 h. The sediment was obtained by


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centrifuging at 10000 rpm for 3 min and washed three times with distilled water and anhydrous ethanol respectively. Finally it was dried by freeze drier. Reaction of FA with other metal ions (Zn2+, Ca2+, Fe2+ and Cu2+) FA (0.5 mmol) and anhydrous ZnCl2 (1 mmol) were added to a mixture of ethanol and H2O with stirring and ultrasonicated for 10 min, followed by addition of N2H4•H2O (85 %) with continuous stirring. The mixture immediately became slurry, then it was transferred to a polytetrafluoroethylene-lined autoclave with heating at 120 °C for 12 h, and cooled naturally to room temperature. The sediment was obtained by centrifuging at 10000 rpm for 3 min and washed three times with distilled water and anhydrous ethanol respectively. Finally it was dried under freeze drier. CaCl2, FeCl2•2H2O and CuCl2 reacted with the FA as the similar method mentioned above. Cell lines and culture conditions The human cervical carcinoma cell (HeLa cell, FR high expression), lung carcinoma cell (A549 cell, FR low expression) and human normal embryonic lung fibroblast cell (L-O2 cell) lines were maintained in DMEM containing 10 % FBS, 100 units/mL penicillin and 100 mg/mL streptomycin in a 5 % CO2 incubator at 37 °C. The cell culture medium was changed every 48 h. Drug loading of FA-Co NTs The loading of anticancer drug DOX was achieved via diffusion effect.9,10,21 Firstly, a calibration curve was plotted in the concentration range of 0.001-1 mg/mL for DOX by diluting the 1 mg/mL standard stock solution of drug in distilled water. The absorbance was measured at 490 nm for DOX against the corresponding solvent blank. The linearity was plotted for absorbance (A) against concentration (C).


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DOX-loaded FA-Co NTs (FA-Co-DOX NTs) were prepared as follows: DOX (40 mg) and FACo NTs (50 mg) were dissolved in distilled water, followed by ultrasonic agitation at a power level of 30 Hz for 2 h, then the mixture was stirred at room temperature for 48 h. The sediment was obtained by centrifuging at 10000 rpm for 3 min and washed three times with distilled water. The supernatant fluid was collected. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: DLC (%) = (weight of loaded drug/weight of NTs) × 100 % 10 DLE (%) = (weight of loaded drug/weight of drug in feed) × 100 % The UV-visible absorption spectra of free DOX and FA-Co-DOX NTs were scanned at 490 nm against the corresponding solvent blank in a 200 µL quartz cuvette and the calibration curve drug loading were measured in triplicate. Drug release of FA-Co-DOX NTs in vitro In vitro DOX release of the FA-Co-DOX NTs was monitored by a UV-vis spectra. The drug release profile was investigated at 37 °C in pH = 5.5 and 7.4 solution and sealed in a dialysis membrane (MWCO = 3500 Dalton). Briefly, 20 mg of FA-Co-DOX NTs were dispersed in 5 mL of different buffer solutions and sealed in the dialysis bag. The dialysis bag was submerged in 100 mL of respective buffer solutions and stirred at 37 °C with gentle shaking. 1 mL portion of the aliquot was collected from the incubation medium at predetermined time intervals (1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h) and replenished with an equal volume of fresh release medium, then the released drug was quantified by UV-vis spectrophotometer at a wavelength of


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490 nm. The total drug content in the released materials was calculated based on the DLE and the amount of drug loaded materials used for the research of release profiles. Cell cytotoxicity assay The cell cytotoxicity of free FA-Co NTs and FA-Co-DOX NTs on HeLa, A549 and L-O2 cells were determined quantitatively by using MTT assay11. All the cells were seeded in 96 well plate at a density of 1 ×104 cells/well and incubated with a series of free FA, CoCl2, DOX, FA-Co NTs and FA-Co-DOX NTs containing the same concentration of DOX for 72 h under the same conditions. The medium was further replaced with fresh culture medium (200 µL) containing MTT solution (20 µL, 5 mg/mL) and then cultured for another 4 h. Finally, MTT containing medium was discarded, and DMSO (150 µL) was used to dissolve formazan crystals. The optical density of the solution was measured by enzyme linked immunosorbent assay (ELISA) at a wavelength of 490 nm. The absorbance value of untreated cells was set at 100 %. Each experiment was repeated three times in triplicate (n = 9). CLSM observation of the cell uptake To investigate the general intracellular distribution of FA-Co NTs, CLSM was utilized to trace the endocytosed FA-Co NTs according to previously reported procedures12,23. 5 ×104 HeLa and L-O2 cells were seeded on a cover slip in a 6 well plate and incubated overnight in a 5 % CO2 incubator at 37 °C for attachment respectively. Cells were then firstly washed with phosphate buffered saline (PBS, pH = 7.4) and treated with FITC, DOX, FITC labelled and DOX loaded nanotubes (10 µg/mL, denoted as FA-Co-DOX-FITC NTs) respectively. After culture for 4 h, the cells were washed with PBS three times and mixed with 500 µL 4 % paraformaldehyde for 20


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min. Subsequently, to further observe cell nuclei, cells were stained with DAPI (2 µg/mL) for 15 min. Cell nuclei and intracellular fluorescent FA-Co NTs were observed by CLSM. Flow cytometry assay To quantify the fraction (percent (%) of FITC positive cells) of cells that internalized FA-Co NTs, flow cytometry was applied. HeLa and L-O2 cells were cultured into a 6 well plate at an initial seeding density of 3 × 104 cells/well. After being treated with FA-Co-FITC NTs (10 µg/mL) at 37 °C for 4 h, cells were collected, washed and re-suspended in PBS. Finally, the percentage of cells internalized with FA-Co NTs and the amount of endocytosed FA-Co NTs inside the cells were analyzed by flow cytometry. 131

I labeling of FA-Co NTs and in vitro stability of the tracer

Iodine-131 (131I), which emits both beta (99 %) and gamma ray (1 %) and has a radioactive halflife of 8.02 days, is frequently used as radiotracers and scintigraphic scanning13,34. As designed, FA-Co complex was the basic framework of FA-Co NTs, here we employed standard Iodogen coating method to oxidize I- ions into I atoms to attack benzene ring in FA molecule via electrophilic substitution reaction to form

131

I-FA-Co NTs according to the reported protocol14.

Briefly, Iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycouril), a water-insoluble oxidant, was dissolved in dichloromethane and coated on the walls of the Eppendorf tube. Radioiodination was then initiated by firstly adding 50 µL DMSO to dissolve Iodogen completely, then followed 400 µL PBS (pH = 7.4) solution of FA-Co NTs (1 mg/mL) and 1 mCi Na131I solution into Iodogen-coated tube. The reaction solution was shaken and incubated at 45 °C for 2 h. Finally, the reaction was terminated by removal of reaction solution from the tube. Excess

131

I was

removed by centrifuging and washing FA-Co NTs by pure water till no detachable gamma


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activity in the supernatant. Radiochemical yield was determined by paper chromatography using Whatman 1 paper strips (eluting solvent: pure water, Rf 131I-FA-Co NTs = 0) and the retained 131I on FA-Co NTs was calculated using the equation (radioactivity on filter/total sampled radioactivity × 100 %). To ensure

131

I is stable on FA-Co NTs as a radiotracer, the stability of the

131

I-FA-Co NTs in

serum was assayed as follows: 100 µL of 131I-FA-Co NTs was added to 900 µL of mouse serum and incubated at 37 °C. After a certain interval, the radiochemical purity of 131I-FA-Co NTs was examined by the same method aforementioned. Biodistribution and pharmacokinetics of 131I-FA-Co NTs All animal experiments were performed in compliance with the local ethics committee. Four to five week-old female Balb/c mice initially weighing 18-20 g were furnished by Experimental Animal Center, Jiangsu Academy of Traditional Chinese Medicine. Free access to food and water was allowed during housing. All mice were randomly distributed into 6 groups (n = 3 per group) and preinjected with cold NaI to block thyroid.

131

I-FA-Co NTs was diluted with saline

and intravenously administered via the tail vein (5 mg/kg of FA-Co NTs corresponding to 10 µCi of

131

I) under anesthesia. The animals were sacrificed and flushed with 10 mL of normal saline

via the heart to clear any blood remaining in organs at various time post-injection (p.i) and the tissues of interest (heart, lung, liver, spleen, stomach (emptied), kidney, small intestine (emptied), large intestine (emptied), bladder, skin, skeleton, muscle and brain) were placed into preweighed scintillation vials and weighed for analysis of 131I activity using the gamma counter. Corrections were made for background radiation and physical decay during counting. Results are expressed as percentage of the injected dose per gram wet tissue (% ID/g) 15,30.


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Pharmacokinetics was investigated using adult male SD rats weighing 200-220 g and the compartmental model was used to calculate the pharmacokinetic parameters for FA-Co NTs by fitting the concentration data in blood using the software WinNonlin. Three rats were intravenously injected with preparation of

131

I-FA-Co NTs (14.8 MBq/kg) through the tail vein

and then 10 µL of blood samples were collected by means of a tail incision at 5 min, 30 min, 1 h, 2 h, 4 h, 12 h and 24 h. Blood samples were measured for radioactive counts as has been stated above and corrections were made for its background radiation and physical decay during counting. Tumor model Three to four week-old Balb/c female nude mice (20-22 g, Nanjing Mu Tu Medical Science and Technology Co.) were inoculated subcutaneously with 1 × 106 HeLa cells (suspended in 100 µL sterile PBS)/mouse in the right side of flank16. Approximate 7 days post-tumor inoculation, the spherical hard lumps could be clearly observed in the injection sites. The tumor sizes were measured every 2 days by a digital caliper. The tumor volume was calculated according to the following equation: volume = tumor width2 × tumor length/217 and the growth curve of tumor was drew. The mice were used for in vivo experiments when the tumor average volume reached 100-200 mm3 (the maximum size and minimum size of tumors were discarded). Tumor targeting study Nine HeLa tumor-bearing mice were randomly divided into three groups and each intravenously injected with 10 µCi of 131I-FA-Co NTs via tail vein. At predetermined time intervials (1 h, 4 h, 24 h), biodistribution studies were carried out to confirm the %ID/g values of major


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organs/tissues (heart, lung, liver, spleen, stomach, kidney and tumor). The radioactivity in these tissues were measured using a gamma counter and presented as % ID/g ± SD. Treatment of tumor-bearing mice To evaluate the curative effects of FA-Co NTs in vivo, nine mice with tumor models were employed, which were divided into three groups. They were intravenously treated with saline (100 µL), DOX (100 µL, 1 mg/mL in saline) and FA-Co NTs (100 µL, 1 mg/mL in saline) per mouse respectively, three times a week. The mice treated with saline were used as the control group. When saline and NTs were injected, the tumor sizes and mice weights were measured every other day. 16 days later, treated mice were sacrificed, and the tumor tissues were removed from the bodies for weighing. ACKNOWLEDGMENT The authors gratefully acknowledge the support of the National Natural Sciences Foundation of China (no. 21401216) and Qing Lan Project in Jiangsu Province. Supporting Information Available IR spectra (Fig S1), zeta potential (Fig S2), TEM image (Fig S3), Table S1, Table S2 and the growth curve of tumor size (Fig S4). Notes The authors declare no competing financial interest.

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An integrative antitumor nanomedicine and tumor-targeted drug carrier formed by FA-Co NTs.


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An Integrative Folate-Based Metal Complex Nanotube as a Potent

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