Anticancer drug delivery to cancer cells using alkyl amine-functionalized nanodiamond supraparticles

Nanocarriers have attracted increasing interest due to their potential applications in anticancer drug delivery. In particular, the ability of nanodiamonds (NDs) to spontaneously self-assemble into unique nano-structured architectures has been exploited in the development of nanocarriers. In this context, we synthesized functional supraparticles (SPs) by the self-assembly of alkyl amine-modified NDs for use in anticancer chemotherapy. The structural, physical, and physiological properties of these ND-SPs as well as their high biocompatibility were assessed using microscopic techniques and various characterization experiments. Finally, a model anticancer drug (CPT; camptothecin) was loaded into the ND-SPs to investigate their anticancer efficacy in vitro and in vivo. After incubation of CPT-loaded ND-SPs with cancer cells, a dramatic anticancer effect of ND-SPs was expressed, compared to CPT-loaded ordinary nanocarriers of polyethylene glycol-modified polymer micelles and conventional Intralipid® 20% emulsions containing CPT. Our results demonstrated that ND-SPs may serve as a nanomedicine with significant therapeutic potential.


Introduction
Various nanomaterials, such as liposomes, polymers, dendrimers, silicon, magnetic nanoparticles, and carbon materials, have been investigated as carriers in targeted anticancer drug delivery systems, using primarily the enhanced permeability and retention (EPR) effect, which is the preferential accumulation of nanoparticles within tumors owing to their leaky vasculature and poor lymphatic drainage. [1][2][3] In particular, nanodiamonds (NDs), which have individual diameters of 2-10 nm and a truncated octahedral structure, have contributed signicantly to the development of highly efficient and successful drug delivery systems due to their signicant potential advantages, such as their relatively low cost, amenability to large-scale synthesis, unique optical properties, characteristic structures, and low toxicity. [4][5][6][7] Self-assembled materials have gained great attention with respect to the design of drug carriers and three-dimensional architectures due to their wide range of applications from controlled drug delivery and tissue engineering to nanoelectronics. 8,9 The ND surface possesses an assortment of functional groups, most of which are oxygenated and amino moieties, including carboxylic acid, lactone, ketone, ether, hydroxyl, and amino groups. These functional groups and/or the hydrophobic surface of NDs have mostly been used as a platform to conjugate with drug molecules through ionic, hydrogen, and hydrophobic interactions. [10][11][12] However, the use of these functional groups as a starting point and a driving force for the self-organization of built up nanoconstructs is still not sufficiently exploited. We believe that these self-assembled formulations of NDs can be further expanded as a modication concept to deliver drugs to site-specic targets. To explore such possibilities, we recently demonstrated that per-uorooctanoic acid (PFOA)-chemically functionalized NDs spontaneously transformed into well-dispersed and biocompatible supraparticle (SP) nanoclusters that could serve as effective drug carriers for cancer treatment. 13 PFOA was used as a model inducer to achieve the SP transformation via strong hydrophobic interactions. However, uorine compounds, including PFOA, have potential environmental and health risks due to their accumulation and retention in biological bodies. 14,15 Therefore, rational and new designs of ND-based SPs are desired for future clinical and biomedical applications.
In this context, we used a variety of alkyl amine derivatives, which have relatively lower biological retention, as alternative materials for PFOA to explore their capacity to form ND-SPs. Different lengths of alkyl chains of amine derivatives were modied onto a carboxylic acid-functionalized ND via simple covalent modication into self-assembled SPs that have wellcontrolled particle sizes. These SPs were further modied with an anticancer drug to investigate the potential improvement of the drug efficacy in comparison to conventional nanocarriers composed of polyethylene glycol (PEG)-modied polymer micelles and an ordinary drug carrier of Intralipid® 20% emulsions.

Characterization of the ND-SPs
The structure and morphology of the prepared ND-SPs were visualized using a high-resolution transmission electron microscope (TEM) (EM-002B, Topcon, Tokyo, Japan) at an accelerating voltage of 120 kV.
A UV-Vis-NIR spectrophotometer (V-730 BIO, Jasco, Tokyo, Japan) was used to measure the spectral proles and concentrations of the ND-SPs and CPT complexes.
Fourier transform infrared (FTIR) spectroscopy (Spectrum One, PerkinElmer, Yokohama, Japan) analysis was carried out to identify the presence of alkyl chains on the ND surface.
Cell viability was assessed using a Cell Counting Kit (CCK)-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Briey, cells were seeded in a 96well plate (5 Â 10 3 cells per well) and allowed to attach overnight. Then, they were exposed to drugs or nanocomplexes as indicated. Aer washing with a fresh medium, the cells were incubated with the CCK-8 solution. A microplate reader (Innite M200 PRO, Tecan, Männedorf, Switzerland) was used to read the absorbance at 450 nm.

In vitro releasing behavior of CPT from ND-SPs
The CPT@Dod-ND-SP powder (5.6 mg) was dispersed in 1 ml of PBS buffer solution (pH 7.4) at 37 C and stirred at 100 rpm. At determined time intervals, 50 ml of the solution was collected and mixed with 200 ml of dimethyl sulfoxide to completely dissolve the drugs. The mixture was then centrifuged at 15 000 rpm for 5 min to remove undissolved ND-SPs. The CPT concentration in the supernatant was nally analyzed using a UV-Vis-NIR spectrophotometer.
Live cell imaging U2OS cells were seeded in imaging dishes the night before treatment. The attached cells were incubated with BODIPYloaded conventional nanocarriers (PEGMEM, F127, and DSPE-PEG) and BODIPY@Dod-ND-SPs for 3 h at 37 C, followed by nuclear staining with Hoechst 33342 (1 mg ml À1 ; Thermo Fisher Scientic, Waltham, MA, USA) for 10 min. Aer three washes with PBS, the cells maintained in the RPMI 1640 phenol red-free medium (Thermo Fisher Scientic) were subjected to live-cell imaging. The images were acquired with a uorescence microscope (IX73, Olympus, Tokyo, Japan) equipped with a mirror unit (IX3-FGFPXL, Olympus) and an electron-multiplying charge-coupled device camera (DP80, Olympus).

In vivo antitumor experiments
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of National Institute of Advanced Industrial Science & Technology (AIST) and experiments were approved by the Animal Ethics Committee of AIST. Female BALB/cAJc-nu/nu mice (6 weeks old, 18 g) were bought from Japan SLC, Inc. (Shizuoka, Japan). Upon arrival, the mice were randomly separated into 3 groups (n ¼ 4), and subcutaneously inoculated in the right ank with HT-29 cells (5 Â 10 5 cells per site) for the construction of the tumor xenogra model. Intraperitoneal injections of 200 ml of Dod-ND-SPs (56 mg kg À1 ), CPT@ND-SPs (CPT, 3 mg kg À1 ; ND-SPs, 56 mg kg À1 ) or PBS were performed every alternate day, when small tumor buds reach about 50 mm 3 . The overall health was monitored by body weight observation and the tumor size was calculated as V ¼ L Â W 2 /2, where L and W are the length and width of the tumor, respectively.

Blood tests
The complete blood count and biochemical parameters were analyzed by Japan SLC, Inc. (Shizuoka, Japan) and Oriental Yeast Co., ltd. (Tokyo, Japan). Briey, 10 week-old female BALB/ cSlc mice (n ¼ 5; average weight ¼ 21 g; Japan SLC) were injected with 200 ml of sterilized water containing ND-SPs (ND-SP ¼ 1.12 mg kg À1 ) or 200 ml of phosphate buffered saline (PBS) buffer via the tail vein. Blood samples were collected from the inferior vena cava of the mice aer 4 weeks.

Statistical analysis
The results are presented as the mean AE standard deviation of at least three independent experiments, with "n" indicating the number of samples per group. Differences between the groups were evaluated using Student's t-test for two groups and two-way analysis of variance for multiple groups. *, **, and *** denote p value is less than 0.05, 0.01, and 0.001, respectively.

Synthesis and characterization of the ND-SPs
To align the effective nanocarrier features of the SPs with alkyl amine molecules, which have a relatively lower bioaccumulation compared to uorine compounds, carboxylic ND (ND-ori)-based SPs (ND-SPs) were designed and synthesized via a condensation reaction between the carboxylic group on the surface of ND-ori and the amino group of the primary alkyl amines, n-octylamine (Oct), dodecylamine (Dod), and oleylamine (Ole), using WSC in an MES buffer (pH 6.0) (Fig. 1). The alkyl chains of the amines immediately trigger the formation of SPs due to their hydrophobic properties. Sonication allows the transformation of the self-assembled SP nanoclusters and the encapsulation of the anticancer drugs into the assembly. The simple preparation of ND-SPs, in addition to the convenient drug loading method, is benecial for various biomedical applications.
Aer the reaction, each of the ND-SP solutions, which have different alkyl chain lengths, showed unique colors and aspects (Fig. 2a). The turbidity of the samples became high due to surface scattering as the length of the alkyl chains increased. 19 Indeed, the absorbances of the ND-SPs increased depending on their apparent turbidity (Fig. 2b). Of the solutions, the spectrum of Ole-ND-SPs showed the highest absorbance due to the suspension. Additionally, the TGA results revealed that approximately 13 wt%, 17 wt%, and 35 wt% of Oct, Dod, and Ole, respectively, were modied onto the ND surface (Fig. 2c). The FTIR peaks, indicated with arrows in Fig. 2D, can be clearly assigned to C-H stretch of the conjugated alkyl amines (Dod), further conrming the formation of ND-SPs.
Due to EPR effects, nano-scale (10-200 nm) particles are favorable for passively targeting tumors. 1-3 Interestingly, according to the DLS measurements, the particle sizes of the prepared ND-SPs (ca. 18-87 nm) make them promising nanocarriers to express EPR effects (Fig. 3a). More interestingly, the diameters of ND-SPs can be easily controlled by changing the length of the alkyl chains. In fact, the DLS proles of Oct-ND-SPs, Dod-ND-SPs, and Ole-ND-SPs displayed well-dened size distributions with different average diameters of 18 nm, 41 nm, 87 nm, respectively (Fig. 3a). The solutions remained stable for at least one week with no obvious change in size. The hydrodynamic diameters of the ND-SPs were higher than those of NDori (ca. 15 nm) due to the self-assembled nanocluster formations. The TEM observations clearly showed that the Fig. 1 Schematic of ND-SP synthesis. Crude ND-ori was functionalized via cross-linking between surface carboxylic groups and the amino groups of alkyl chains via a condensation reaction followed by sonication to promote the self-assembly of ND-SP nanoclusters.
synthesized ND-SPs had cluster nanostructures based on the building blocks of ND-ori (Fig. 3b). The size of each ND-SP, estimated via TEM observations, depicted the same order of particle size as the DLS measurements. These results clearly indicate that alkyl amines can induce self-assembled SP formation of NDs via simple chemical functionalization.
Next, the cytotoxicity of the ND-SPs was investigated because this is a very important issue for future clinical and biomedical applications. CCK-8 assays were used to analyze the survival of the U2OS cells (Fig. 4a). The U2OS cells were pre-incubated with ve different ND concentrations (ND ¼ 0 mg ml À1 , 7 mg ml À1 , 14 mg ml À1 , 28 mg ml À1 , and 56 mg ml À1 ) using the three types of ND-SPs. Over 98% of the cells were viable following the treatment with Oct-ND-SPs or Dod-ND-SPs at all concentrations. Additionally, Ole-ND-SPs displayed a higher cytotoxicity of more than 14 mg ml À1 of ND, likely due to the excess and stronger interaction between the cell surface and the longer alkyl chains of Ole, resulting in the denaturation of the cell membrane. Accordingly, Oct-ND-SPs and Dod-ND-SPs were used for further drug efficacy tests due to their low cytotoxicity.
The drug CPT has a wide range of antitumor effects on cancers. 20 CPT-based drugs are specic inhibitors of topoisomerase 1, leading to the destruction of DNA, and are currently being used as useful chemotherapeutic agents in clinical antitumor treatments. The CPT molecules were encapsulated in the ND-SPs via simple sonication, as shown in Fig. 1. The UV-Vis-NIR spectra displayed characteristic peaks of CPT at 350 nm and 369 nm aer encapsulation (Fig. 4b). To test the anticancer drug efficacy of the functionalized ND-SPs, we incubated the U2OS cells with CPT-loaded ND-SPs [CPT@Oct-ND-SPs or CPT@Dod-ND-SPs] or CPT-loaded ND-ori (CPT@ND-ori) for 24 h. Aer washing with a fresh growth medium, the cell viability was analyzed using a CCK-8 kit. CPT@Dod-ND-SPs showed the highest anticancer chemotherapeutic effect (ca. 49%) at 10 mg ml À1 of CPT compared to the maximum drug efficacy values of the other materials [CPT@Oct-ND-SPs at $39% and CPT@ND-ori at $36%] (Fig. 4c). Interestingly, CPT@Dod-ND-SPs did not exhibit strong drug efficacy on TIG-3 and MRC5 normal broblast cell lines in comparison with U2OS osteosarcoma cells because of its potential targeting effect (Fig. S1a †). Besides, Dod-ND-SPs themselves do not have   any cytotoxicity against these broblast cell lines (Fig. S1b †). More surprisingly, the most dramatic decrease in cell viability (down to 10%) was caused by increasing the CPT concentration to 20 mg ml À1 , where CPT@DOD-ND-SPs exhibited better anticancer efficacy than conventional nanocarriers such as PEG-MEM-, F127-, and DSPE-PEG-based polymer micelles including CPT molecules. It is well known that carbon-based nanomaterials have high biological affinities against cells. [21][22][23] Additionally, the lower drug efficacies of conventional nanocarriers are likely due to PEG moieties on their constructs that generally inhibit interactions with cells. 24,25 To clarify the effective transmembrane permeation properties of ND-SPs, uorescence live cell imaging was performed (Fig. 5). A hydrophobic uorescent molecule (BODIPY) was co-assembled with ND-SPs via noncovalent hydrophobic interaction. Fluorescence microscopy showed that compared to PEGMEM-, F127-, and DSPE-PEGbased polymer micelles, Dod-ND-SPs were more efficiently internalized by the U2OS cells at 37 C. Furthermore, the efficacy of CPT suspended in Intralipid® 20% emulsion, which is one of the recommended drug carriers, was also compared with the anticancer effect of CPT@Dod-ND-SPs (Fig. S2 †). Dod-ND-SPs showed a stronger drug efficacy than Intralipid® 20%. These results clearly indicate that drug-loaded functional ND-SPs can effectively eliminate cancer cells.
The nal goal of this research is to build a functional drug delivery system of ND-SPs. Herein, we have calculated the loading capacity and efficiency (Fig. S3a †). In vitro release proles of CPT molecules from Dod-ND-SPs were monitored in PBS buffer (pH 7.4) (Fig. S3b †). CPT molecules were rapidly released from Dod-ND-SPs over time, where it reached a plateau aer about 24 h, and were released up to 75% in PBS aer 72 h.
To demonstrate the feasibility of CPT-loaded ND-SPs for cancer treatment in vivo, the antitumor activity of CPT@Dod-ND-SPs was evaluated using HT-29 tumor xenogra models. As shown in Fig. 6A, the tumor growth was strongly suppressed in the CPT@Dod-ND-SP treatment group as compared to the PBS control, although Dod-ND-SPs alone didn't show signicant difference. At the end of treatment, the tumor size of the mice receiving CPT@Dod-ND-SPs was remarkably smaller than those receiving PBS (Fig. 6B), suggesting that CPT@Dod-ND-SPs can retard tumor progression effectively. In addition, there was no signicant loss of body weight in the mice (Fig. 6C), demonstrating that the systemic toxicity of CPT@Dod-ND-SPs is negligible.
To investigate the biocompatibility of ND-SPs further, mice were intravenously administered with sterilized water containing Dod-ND-SPs (200 ml; Dod-ND-SPs, 1.12 mg kg À1 ) or 200 ml of the PBS buffer for 4 weeks and subsequent blood tests were performed (Tables 1 and 2). The hematological and biochemical   parameters did not differ between the mice intravenously injected with Dod-ND-SPs and PBS, conrming the absence of an inammatory response or systemic side effects and underscoring the biocompatibility of ND-SPs.

Conclusions
We demonstrated the spontaneous self-assembly of commercially available alkyl amine-modied NDs into unique geometrical architectures leading to the formation of SPs for the future treatment of osteosarcoma. We conducted a detailed investigation of SP formation for different lengths of alkyl chains of the primary amines. The structural, physical, and physiological properties of these ND-SPs were assessed using microscopic techniques and optical, thermal gravimetric, and cell tests. The sizes of the ND-SPs were easily controlled by changing the length of the alkyl chains of the amines. A model anticancer drug CPT was incorporated into these ND-SPs and conventional PEG-modied polymer micelles, and the ND-SPs showed good drug efficacy against U2OS bone osteosarcoma cells. In particular, CPT-loaded ND-SPs displayed the highest anticancer therapeutic effect. In vivo anti-tumor assays showed that CPT delivered by ND-SPs caused stronger tumor suppression of HT-29 colorectal adenocarcinoma xenogras. These self-assembled ND-SPs open up possibilities for various applications and will be explored further for drug delivery. Compared to ordinary nanocarriers, ND-based materials generally possess the overwhelming superiorities of easy size control, robustness against chemicals and physical conditions, good thermal stability, and unique optical properties. 26,27 However, the investigation of ND-SPs as drug carriers is still in its infancy. In this regard, developing ND-SPs is highly promising for various clinical and biological applications for cancer therapy.

Conflicts of interest
There are no conicts to declare.