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. Author manuscript; available in PMC: 2020 Oct 30.
Published in final edited form as: Int J Pharm. 2019 Aug 12;570:118609. doi: 10.1016/j.ijpharm.2019.118609

A solid lipid nanoparticle formulation of 4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine with increased solubility, stability, and antitumor activity

Solange A Valdes 1, Riyad F Alzhrani 1, Andres Rodriguez 2, Dharmika SP Lansakara-P 1, Sachin G Thakkar 1, Zhengrong Cui 1
PMCID: PMC6824728  NIHMSID: NIHMS1539153  PMID: 31415878

Abstract

Previously, we synthesized 4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine (DHA-dFdC), a novel lipophilic compound with a potent, broad-spectrum antitumor activity. Herein, we report a solid lipid nanoparticle (SLN) formulation of DHA-dFdC with improved apparent aqueous solubility, chemical stability, as well as efficacy in a mouse model. The SLNs were prepared from lecithin/glycerol monostearate-in-water emulsions emulsified with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and Tween 20. The resultant DHA-dFdC-SLNs were 102.2 ± 7.3 nm in diameter and increased the apparent solubility of DHA-dFdC in water to at least 5.2 mg/mL, more than 200-fold higher than its intrinsic water solubility. DHA-dFdC in a lyophilized powder of DHA-dFdC-SLNs was significantly more stable than the waxy solid of pure DHA-dFdC. DHA-dFdC-SLNs also showed an increased cytotoxicity against certain tumor cells than DHA-dFdC. The plasma concentration of DHA-dFdC in mice intravenously injected with DHA-dFdC-SLNs in dispersion followed a bi-exponential model, with a half-life of ~44 h. In mice bearing B16-F10 murine melanoma, DHA-dFdC-SLNs were significantly more effective than DHA-dFdC in controlling the tumor growth. In addition, histology evaluation revealed a high level of apoptosis and tumor encapsulation in tumors in mice treated with DHA-dFdC-SLNs. DHA-dFdC-SLNs represents a new DHA-dFdC formulation with improved antitumor activity.

Keywords: Solid lipid nanoparticles, solubility, stability, antitumor activity, cytotoxicity, plasma pharmacokinetics

1. Introduction

Previously we synthesized DHA-dFdC (Fig. 1A) by conjugating docosahexaenoic acid (DHA), a omega-3 polyunsaturated fatty acid (PUFA), to gemcitabine (2′, 2-difluorodeoxycytidine, dFdC) on its 4-N position (Naguib et al., 2016). DHA-dFdC showed potent and broad-spectrum antitumor activity against the National Cancer Institute (NCI)-60 human tumor cell lines and was significantly more effective than the molar equivalent dose of gemcitabine in controlling tumor growth in several mouse models of pancreatic cancer, including a genetically engineered mouse model and athymic mice with orthotopically transplanted human pancreatic tumor cells (Naguib et al., 2016). However, DHA-dFdC is poorly soluble in water (i.e. intrinsic solubility, ~25 μg/mL), and a formulation in which DHA-dFdC is chemically more stable than in the current Tween 80-ethanol-in-water formulation is desired (Naguib et al., 2016).

Figure 1.

Figure 1.

Figure 1.

Figure 1.

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(A) Chemical structure of DHA-dFdC. (B-D) Effect of the amount of DHA-dFdC used to prepare the DHA-dFdC-SLNs on the particle size (B), polydispersity index (C), and zeta potential (D) of the resultant DHA-dFdC-SLNs after 6 days of storage at 4°C. Data shown are mean ± SD (n = 3). (E) Particle size distribution curves of three independently prepared DHA-dFdC-SLNs that were prepared with 5.2 mg of DHA-dFdC. (F) A representative TEM image of DHA-dFdC-SLNs prepared with 5.2 mg of DHA-dFdC (bar = 200 nm). (G) A presentative gel permeation chromatograph of DHA-dFdC-SLNs prepared with 5.2 mg of DHA-dFdC. DHA-dFdC-SLNs were applied to a Sepharose 4B column, and the elution fraction was 0.5 mL. In G, the experiment was repeated at least three times with similar results.

SLNs have emerged as an attractive delivery system for poorly water-soluble drugs (Feng and Mumper, 2013; Geszke-Moritz and Moritz, 2016; MuÈller et al., 2000). There is also evidence that incorporation of a drug into SLNs can increase its chemical stability (Lim et al., 2004; Patel et al., 2012; Üner et al., 2005). Previously, our group developed a SLN formulation based on a lecithin/glycerol monostearate (GMS)-in-water emulsion (Sloat et al., 2010; Sloat et al., 2011; Wang et al., 2017). In the present study, we developed a new DHA-dFdC SLN formulation (DHA-dFdC-SLNs) by incorporating DHA-dFdC into SLNs prepared by emulsifying lecithin/GMS-in-water emulsions with Tween 20 and TPGS (or vitamin E TPGS) (Sloat et al., 2010; Sloat et al., 2011). TPGS is a water-soluble derivate of natural vitamin E, which is formed by esterification of vitamin E succinate with polyethylene glycol (PEG) (Zhang et al., 2012). TPGS is widely used in pharmaceutical formulations as an emulsifier, solubilizer, absorption enhancer, permeation enhancer, and/or stabilizer (Cho et al., 2014; Mu and Feng, 2002; Muthu et al., 2011; Zhang et al., 2012). There is also evidence that TPGS has a stronger antioxidant activity than α-tocopherol or vitamin E (Anstee et al., 2010; Carini et al., 1990), which is expected to be beneficial as DHA-dFdC is sensitive to oxidation, and data from our previous studies showed that vitamin E as an antioxidant can help to stabilize DHA-dFdC (Naguib et al., 2016). Moreover, data from several studies showed that TPGS induces apoptosis and has a synergic effect with certain cancer chemotherapeutic agents such as docetaxel, paclitaxel, and doxorubicin (Assanhou et al., 2015; Mi et al., 2011; Youk et al., 2005; Yu et al., 2015; Zhu et al., 2014). The new DHA-dFdC-SLN formulation significantly increased the apparent water solubility and chemical stability of DHA-dFdC. We also evaluated the plasma pharmacokinetics of DHA-dFdC in a mouse model after DHA-dFdC was administered by intravenous injection of the DHA-dFdC-SLNs in dispersion, the cytotoxicity of the DHA-dFdC-SLNs against several tumor cell lines in culture, and the antitumor activity of the DHA-dFdC-SLNs in a mouse model with B16-F10 murine melanoma cells.

2. Materials and methods

2.1. Materials and cell lines

Mannitol, Tween 20, GMS, TPGS1k, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tween 80, mannitol, and sucrose were from Sigma-Aldrich (St. Louis, MO). Gemcitabine HCl was from Biotang, Inc. (Lexington, MA). Soy lecithin (refined) was from Alfa Aesar (Ward Hill, MA). Ethyl acetate, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), isopropanol, and methanol were from Thermo Fisher (Waltham, MA). Float-A-Lyzer®G2 dialysis device (molecular weight cutoff, 50 kDa) was from Spectrum Chemicals & Laboratory Products (New Brunswick, NJ).

B16-F10 murine melanoma cell and TC-1 murine lung cancer cell lines were from the American Type Culture Collection (Manassas, VA). M-Wnt murine mammary gland cells were from Dr. Stephen D. Hursting’s lab at The University of North Carolina, Chapel Hill. B16-F10 and TC-1 cells were grown in Dulbecco’s Modified Eagle Media (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 media, respectively (Invitrogen, Carlsbad, CA). M-Wnt cells were grown in a similar medium as TC-1, with an additional supplement of 1% Glutamax (GlutaMAX™Supplement, Gibco®). All media were supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin, all from Invitrogen-Life Technologies (Carlsbad, CA).

2.2. Preparation and characterization of 4-(N)-docosahexaenoyl 2′,2′-difluorodeoxycytidine solid lipid nanoparticles (DHA-dFdC-SLNs)

2.2.1. Preparation of DHA-dFdC-SLNs

DHA-dFdC was synthesized following our previously reported conjugation scheme (Naguib et al., 2016). The purity of the resultant DHA-dFdC was confirmed by Nuclear Magnetic Resonance (NMR) Spectroscopy and Mass Spectrometry. SLNs were prepared as previously described with some modifications (Sloat et al., 2010). Briefly, 3.5 mg of soy lecithin, 0.5 mg of GMS, and 0.875 mg of TPGS were weighed into a 7 mL glass vial. Eight hundred microliter of de-ionized and filtered (0.22 μm) water (80°C) was added into the lecithin/GMS/TPGS mixture, which was then vortexed and sonicated for 3 min until a homogenous slurry was formed. The mixture was maintained on an 80°C hot plate surface for 5 min. A solution of Tween 20 (200 mcL) was then added drop wise into the mixture to reach a final concentration of 1% (v/v). The resultant emulsions were allowed to cool to room temperature while stirring to form SLNs. To incorporate DHA-dFdC into the SLNs, HA-dFdC at various amounts (i.e. 5.2, 8.3, 9.8, or 14.3 mg) was added into the lecithin/GMS/TPGS mixture before the addition of water. The steps followed were identical to the preparation of DHA-dFdC-free SLNs. The size and zeta potential of the SLNs were measured using a Malvern Zetasizer Nano ZS (Westborough, MA).

2.2.2. Short-term stability study

The stability of DHA-dFdC-SLNs prepared with 0, 5.2, 8.3, 9.8, or 14.3 mg of DHA-dFdC was evaluated at 4°C for 6 days in order to identify the most stable formulation for further studies. The size and zeta potential of the SLNs were measured using a Malvern Zetasizer Nano ZS (Westborough, MA).

2.2.3. Transmission electron microscopy (TEM)

The size and morphology of the DHA-dFdC-SLNs were examined using a transmission electron microscope as previously described (Zhu et al., 2012).

2.2.4. Encapsulation efficiency

The encapsulation efficiency of DHA-dFdC in SLNs was determined by an ultrafiltration method. Briefly, 1 mL of DHA-dFdC-SLNs in dispersion was added into an ultrafiltration centrifuge tube (30 kDa, Amicon Ultra-4, Millipore) and centrifuged at 2844 relative centrifugal force (rcf) for 10 min. Then, 100 μl of the filtrate solution was taken from the bottom part of the ultrafiltration centrifuge tube to measure DHA-dFdC concentration by high performance liquid chromatography (HPLC). To corroborate the detection method, the remaining suspension (50 μl) in the ultrafiltration centrifuge tube was diluted with 950 μl water to extract the DHA-dFdC, according the procedure previously described (Sloat et al., 2011), and the content of DHA-dFdC was determined using HPLC.

2.2.5. Gel permeation chromatography (GPC)

To separate micelles from DHA-dFdC-SLNs, GPC was performed using a 6 mm × 30 cm Sepharose® 4B column (Sloat et al., 2011). Samples (100 μL) were applied into the column and eluted with de-ionized and filtered (0.22 μm) water. Elution fractions of 500 μL were collected. Particle concentration (i.e. kilocounts per second or Kcps) in each fraction was measured using a Malvern Zetasizer Nano ZS, and the concentration of DHA-dFdC in each fraction was determined using HPLC after extraction.

2.3. Lyophilization of the DHA-dFdC-SLNs and their stability in lyophilized powder

A 30% (w/v) stock solution of sucrose as lyoprotectant was prepared with de-ionized and filtered (0.2 μm) water. Then, 900 μL of DHA-dFdC-SLNs in water was mixed with 100 μL of the sucrose solution to obtain a final suspension with 3% (w/v) of sucrose. The DHA-dFdC-SLNs in suspension were then stored at −20°C for 30 min, transferred to −80°C for 60 min, and finally transferred to a VirTis Advantage bench top tray lyophilizer (The VirTis Company, Inc. Gardiner, NY). Lyophilization was performed over 72 h at a pressure of less than 200 mTorr. The shelf temperature was gradually ramped from −40°C to 26°C. After lyophilization, the sample was quickly sealed and stored in a desiccator at room temperature (~24°C), protected from light. To evaluate the physical and chemical stability of the DHA-dFdC-SLNs in the lyophilized powder, DHA-dFdC was extracted from the powder 0, 7, and 30 days later. Briefly, the lyophilized sample was reconstituted in 1 mL of de-ionized and filtered (0.2 μm) water. The reconstituted DHA-dFdC-SLNs in suspension (100 μL) were mixed with 100 μL of isopropanol, vortexed for 30 s, and maintained at room temperature for 5 min. Then, 600 μl of ethyl acetate was added, and the sample was vortexed for 30 s and centrifuged at 11,000 rcf for 20 min. The supernatant was collected into a glass vial and evaporated under nitrogen. The sample was re-dissolved in 100 μL of THF, and the concentration was measured by HPLC.

As a control, DHA-dFdC was dissolved in ethanol and then mixed with vitamin E at final concentration of 5.047% (w/w) (Naguib et al., 2016). The solution was dried under nitrogen, sealed, and stored at room temperature, protected from light, and the content of DHA-dFdC was measured at various time points.

2.4. In vitro stability of DHA-dFdC-SLNs in simulated biological media

To confirm that the DHA-dFdC-SLNs do not aggregate in the presence of serum albumin, the SLNs in suspension were diluted in phosphate-buffered saline (PBS, 10 mM, pH 7.4) with 10% FBS (v/v) and incubated at 37°C in a MaxQ 4000 Floor Shaker Incubator (Thermo Fisher Scientific, 100 revolutions per min (rpm)) for 18 h. The particle size was measured at different time points using a Malvern Zetasizer.

2.5. In vitro release of DHA-dFdC from DHA-dFdC-SLNs

The release profile of DHA-dFdC from SLNs were evaluated by suspending DHA-dFdC-SLNs at a concentration of 127 μg of DHA-dFdC per mL in release medium (i.e. PBS with 1% (w/v) of Tween 20), which were then placed into a 1 mL cellulose ester dialysis tube (molecular weight cutoff, 50,000). The dialysis tube was then placed into a plastic conical tube containing 13 mL of release medium to create sink conditions, which was maintained in a MaxQ 5000 Floor Shaker Incubator at 37°C and 100 rpm for 8 h. At predetermined time points, 200 μL of the release medium was withdrawn and replaced with 200 μL of fresh release medium. As a control, the diffusion of DHA-dFdC dissolved in a Tween 20 solution (i.e. 127 μL g/mL of DHA-dFdC in 1% of Tween 20 in water) across the dialysis membrane was also measured. The concentration of the DHA-dFdC was determined by HPLC.

2.6. HPLC

HPLC analysis of DHA-dFdC was performed using an Agilent Infinity 1260 (Santa Clara, CA) with a RP-C18 column (Zorbax Eclipse, 5 μm, 4.5 mm × 150 mm, Santa Clara, CA). The mobile phase was methanol and water (90:10, v/v). The flow rate was 1.0 mL/min, and the detection wavelength and injection volume were 248 nm and 5 μL, respectively (Naguib et al., 2016).

2.7. In vitro cytotoxicity assay

The cytotoxicity of DHA-dFdC-SLNs was evaluated in TC-1, B16-F10, and M-Wnt cells. Cells were seeded into 96-well plates (4,000 cells/well for TC-1 and B16-F10 cells, 1,000 cells/well for M-Wnt cells) and incubated at 37°C, 5% CO2 overnight. Cells were then treated with various concentrations of DHA-dFdC, DHA-dFdC-SLNs, or DHA-dFdC-free SLNs for up to 48 h. As controls, cells were treated with fresh media or media containing DMSO. Cell survival was determined using an MTT assay (Naguib et al., 2014). DHA-dFdC was dissolved in DMSO and then diluted with cell culture media, whereas DHA-dFdC-SLNs and DHA-dFdC-free SLNs were dispersed directly in cell culture media.

2.8. Pharmacokinetics (PK) of DHA-dFdC

The animal protocol was approved by the Institutional Animal Care and Use Committee at The University of Texas at Austin. To evaluate the PK parameters, healthy female C57BL/6 mice (6–8 weeks, Charles River Laboratories, Wilmington, MA) were injected intravenously (i.v.) with DHA-dFdC-SLNs dispersed in sterile mannitol 5% (w/v) at dose of 2 mg of DHA-dFdC per mouse. Mice were euthanized at various time points later (i.e. 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h). Blood was collected into heparin-coated tubes, which were then centrifuged at 13000 rcf for 20 min to isolate plasma. Then 200 μL of the plasma was mixed with 200 μL of isopropanol and 200 μL cold PBS. The mixture was vortexed and incubated at 4°C for 5 min. Following the incubation, 1000 μL of ethyl acetate was added, and the mixture was vortexed for 5 min, and followed by centrifugation at 18,000 rcf for 5 min. The supernatant was collected and dried under nitrogen gas. Finally, the residue was re-dissolved in 100 μL of THF, which was then analyzed using HPLC to measure DHA-dFdC concentration (Naguib et al., 2016). As an internal control, 4-(N)-stearoyl dFdC synthesized by conjugating stearate to dFdC on its 4-(N) position was added in the samples before extraction (Sloat et al., 2011). Data were analyzed using the PK Solver®, assuming a two-compartmental model (Zhang et al., 2010).

2.9. Evaluation of the antitumor activity of DHA-dFdC-SLNs in a mouse model

Female C57BL/6 mice (18–20 g, 6–8 weeks) were subcutaneously (s.c.) injected with B16-F10 (5 × 105 cells/mouse) in the right flank on day 0. Seven days later, mice were randomized in 5 groups (n = 5–6) and i.v. injected with DHA-dFdC (50 mg/kg) dissolved in a vehicle solution (i.e. Tween 80 (10%, w/v), ethanol (5.2%, v/v), and mannitol (5%, w/v) in water), the vehicle solution (as a control) (Naguib et al., 2016; Valdes et al., 2017), DHA-dFdC-SLNs (equivalent to 50 mg/kg of DHA-dFdC), or the equivalent dose of DHA-dFdC-free SLNs; both SLNs were dispersed in sterile mannitol 5%, (w/v). As a control, one group of mice were left untreated. Treatments were repeated every 3 days for a total of 4 times. Mouse health and tumor growth were monitored daily. Tumor size was measured 2–3 times a week, and tumor volume was calculated as: volume (mm3) = (length × width2)/2. Mice were euthanized 17 days after B16-F10 cell injection, and tumor tissues were collected for histology study. For mice that were left untreated, the length of some of the tumors reached 15 mm before day 17 and had to be euthanized earlier.

2.10. Histology

Tumor tissues were fixed in formalin, embedded, and stained with hematoxylin and eosin (H&E) in the Histological and Tissue Analysis Facility in the Dell Pediatric Research Institute at The University of Texas at Austin.

2.11. Data analysis

Statistical analyses were completed by one-way analysis of variance followed by a Bonferroni post hoc test. A p value of ≤ 0.05 (two-tail) was considered significant. Most of the analyses were performed with GraphPad Prism (GraphPad Software, Inc., La Jolla, CA).

3. Results and discussion

3.1. Preparation and characterization of DHA-dFdC-SLNs

DHA-dFdC is a lipophilic compound with potent antitumor activity against various cancer cell lines in culture (e.g. pancreatic cancer, leukemia, kidney cancer) and in mouse models of pancreatic cancer and leukemia (Naguib et al., 2016; Valdes et al., 2017). However, this compound presents solubility and stability challenges (Naguib et al., 2016). To increase its water solubility and chemical stability, we incorporated it into a SLN formulation prepared with lecithin, GMS, TPGS, and Tween 20.

The particle diameter, polydispersity index, and zeta potential of DHA-dFdC-SLNs prepared with various concentrations/amounts of DHA-dFdC are shown in Table 1. Statistical analysis did not reveal any significant differences in the particle sizes and zeta potentials of SLNs prepared with various amounts of DHA-dFdC. However, in a short-term stability study at 4°C, the DHA-dFdC-SLNs prepared with 5.2 mg of DHA-dFdC remained stable after 6 days (Fig. 1B–D) and were thus selected for further studies. The SLNs increased the apparent aqueous solubility of DHA-dFdC to 5.2 mg/mL, which may be further increased by concentrating the nanoparticles. Shown in Fig. 1E is the dynamic light scattering spectrum of DHA-dFdC-SLNs prepared with 5.2 mg of DHA-dFdC, showing that the DHA-dFdC-SLNs were around 100 nm in diameter. The TEM images of the DHA-dFdC-SLNs showed that nanoparticles were spherical (Fig. 1F), with particle size smaller than that determined by dynamic light scattering (Fig. 1E, Table 1), which is expected. The encapsulation efficiency of DHA-dFdC in the DHA-dFdC-SLNs was close to 100% since DHA-dFdC was not detected in the filtrate after ultrafiltration. To corroborate this result, the suspension remained in the ultrafiltration centrifuge tube was re-dissolved in water to extract the DHA-dFdC, and 97% ± 21.4 (n = 6) of DHA-dFdC was recovered. It is possible that the TPGS may have helped to increase the encapsulation efficacy of the DHA-dFdC in the SLNs, because there are reports that TPGS as an emulsifier in paclitaxel-loaded polymeric nanoparticles helped to improve the encapsulation efficiency of paclitaxel to 100% (Mu and Feng, 2002; Mu and Feng, 2003; Zhang et al., 2012). Due to the presence of Tween 20 and TPGS in our DHA-dFdC-SLN formulation, it is possible that a certain fraction of the DHA-dFdC may be in micelles. For instance, TPGS has a relative low critical micelle concentration of 0.02% (w/w) at 37°C, ~ 1% (w/v) for Tween 20 at 20°C (Kim and Hsieh, 2001; Wu and Hopkins, 1999). Therefore, GPC was applied to identify the extent to which DHA-dFdC was potentially incorporated into micelles (Sloat et al., 2011). As shown in Fig. 1G, only one apparent DHA-dFdC elution peak can be identified in the GPC spectrum, which also overlaps with particle count spectrum, providing additional evidence that all the DHA-dFdC was encapsulated into the DHA-dFdC-SLNs.

Table 1.

Characterization of DHA-dFdC-SLNs prepared with different concentrations of DHA-dFdC, while the concentrations of lecithin, GMS, and TPGS were kept at 3.5 mg/mL, 0.5 mg/mL, and 0.875 mg/mL, respectively, and the final concentration of Tween 20 was kept at 1% (v/v). Data shown are mean ± S.D. (n = 3).

DHA-dFdC (mg/mL) 0 5.2 8.3 9.8
Particle diameter (nm) 97.3 ± 13.6 102.2 ± 7.3 92.0 ± 3.6 96.5 ± 14.2
Polydispersity index 0.27 ± 0.10 0.23 ± 0.01 0.24 ± 0.02 0.26 ± 0.02
Zeta potential (mV) −51.5 ± 0.1 −55.3 ± 3.0 −60.7 ± 2.4 −57.7 ± 2.9
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3.2. Chemical stability of DHA-dFdC in DHA-dFdC-SLNs after lyophilization

The DHA-dFdC-SLNs were first lyophilized into a dry powder before studying its stability. To select a proper lyoprotectant, we tested sucrose, mannitol, and trehalose with concentrations ranging from 2.5% (w/v) to 5% (w/v) and found that sucrose at concentrations between 2.5% to 3% can effectively prevent particle size change after the DHA-dFdC-SLNs were subjected to lyophilization and reconstitution (data not shown). Sucrose at 3% (w/v) was thus used as the lyoprotectant for further studies. As shown in Fig. 2A, the particle size of the DHA-dFdC-SLNs did not change significantly after 30 days of storage as a lyophilized powder at room temperature. Importantly, the content of DHA-dFdC in the lyophilized DHA-dFdC-SLNs powder remained unchanged during the 30 days of storage (Fig. 2B). As a comparison, just 19.1% ± 7.0 of DHA-dFdC was let in the DHA-dFdC-vitamin E solid mixture was left after 14 days of storage in the same condition (p < 0.0001) (Fig. 2C). Previously, we reported that DHA-dFdC in a Tween 80-ethanol-water solution was unstable when storage at room temperature, with a half-life of ~14 h, while the addition of vitamin E (0.01%, w/v) in the solution helped to significantly increase the stability of DHA-dFdC in the solution (Naguib et al., 2016). The improved chemical stability of DHA-dFdC in the DHA-dFdC-SLNs dry powder may be attributed to the following three reasons. First, the SLNs may have protected DHA-dFdC incorporated in them from chemical degradation (Geszke-Moritz and Moritz, 2016). For example, it was reported that β-carotene loaded in SLNs have improved stability because the β-carotene was protected against oxidation (Geszke-Moritz and Moritz, 2016; Yi et al., 2014). Second, the TPGS in the formulation likely provided antioxidant properties since TPGS contains α-tocopherol and vitamin E and was reported to have more antioxidant activity than free α-tocopherol and vitamin E (Anstee et al., 2010; Carini et al., 1990). Third, the SLNs were lyophilized into a dry powder (do Vale Morais et al., 2016; Varshosaz et al., 2012; Vighi et al., 2007).

Figure 2.

Figure 2.

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Stability of DHA-dFdC and DHA-dFdC-SLNs as a lyophilized powder. (A) Particle size of DHA-dFdC-SLNs. (B) The concentration of DHA-dFdC remaining in the DHA-dFdC-SLNs. The measures in A and B were performed on 0, 7 and 30 days after the DHA-dFdC-SLNs were lyophilized and stored at room temperature. (C) Chemical stability of DHA-dFdC in a dry waxy solid that contains 5.047% (w/w) of vitamin E when stored at room temperature for 14 days. *** p < 0.001. Data shown are mean ± S.D. (n = 3).

3.3. In vitro characterization of DHA-dFdC-SLNs

The particles size of DHA-dFdC-SLNs after 18 h of incubation in a simulated biological medium (i.e. 10% FBS in PBS) at 37° C did not increase (data not shown), suggesting that DHA-dFdC-SLNs will not likely aggregate in blood circulation after intravenous administration. Shown in Fig. 3 is the release profile of DHA-dFdC from the DHA-dFdC-SLNs. Only 8.6% ± 1.9 of DHA-dFdC was released from the SLNs within 8 h.

Figure 3.

Figure 3.

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In vitro release profile of DHA-dFdC from DHA-dFdC-SLNs. The diffusion of DHA-dFdC (in Tween 20 micelles) across the dialysis membrane was also measured. Data shown are mean ± SD (n = 3).

3.4. Evaluation of the cytotoxicity of the DHA-dFdC-SLNs against tumor cells in culture

The cytotoxicity of the DHA-dFdC-SLNs was evaluated by determining the survival of tumor cells after incubation with the SLNs. As shown in Fig. 4A–B, DHA-dFdC-SLNs were more cytotoxic than DHA-dFdC in M-Wnt (i.e. IC50 values of 0.92 μM versus 2.15 μM, p < 0.05, 24 h of incubation) and B16-F10 cells (i.e. IC50 values of 0.085 μM versus 1.81 μM, p < 0.0001, 48 h of incubation). In TC-1 cells, the cytotoxicity of DHA-dFdC-SLNs was not significantly different from that of DHA-dFdC (Fig. 4C). DHA-dFdC-free SLNs and DMSO (i.e. vehicle used to dissolve DHA-dFdC) did not show significant cytotoxicity in the concentrations tested in all three cell lines (Fig. 4).

Figure 4.

Figure 4.

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Cytotoxicity of DHA-dFdC-SLNs in M-Wnt cells (A), B16-F10 cells (B), and TC-1 cells (C). In A, the cells were incubated with the nanoparticles for 24 h, 48 h for B and C. As controls, cells were also incubated with DHA-dFdC-free SLNs, DHA-dFdC dissolved in DMSO, the equivalent concentration of DMSO, or cell culture media alone. Data shown are mean ± SD (n > 3).

Data from previous studies support that TPGS improve the activity of nanoparticles by enhancing cell uptake and/or increasing cytotoxicity (Li et al., 2016; Muthu et al., 2011; Zhang et al., 2012). For example, high cellular uptake and cytotoxicity of docetaxel were reported when docetaxel-loaded liposomes were coated with TPGS, as compared to PEGylated liposomes without TPGS (Muthu et al., 2011). Other TPGS-emulsified nanoparticles or TPGS-based nanoparticles were also showed to have high cellular uptake and cytotoxicity in cell lines such as Caco-2, HT-29, MCF-7, C6 gliomas cells (Zhang et al., 2012). In addition, data from some studies showed that there is a synergic effect between TPGS and docetaxel, doxorubicin, and paclitaxel (Assanhou et al., 2015; Mi et al., 2011; Zhu et al., 2014). For DHA-dFdC, we have reported that it has potent cytotoxicity against many tumor cells, including the TC-1 cell, which explains that incorporating it into SLNs may not necessarily further improve its cytotoxicity against some cell lines such as the TC-1 cells (Naguib et al., 2016).

3.5. Pharmacokinetic (PK) of DHA-dFdC

Data in Fig. 5 showed the DHA-dFdC levels in mouse plasma samples at different time points after intravenous injection of DHA-dFdC-SLNs. The elimination of DHA-dFdC in mouse plasma follows a bi-exponential model, an initial deposition phase followed by a terminal elimination phase. Table 2 includes selected PK parameters of DHA-dFdC. The AUC0–∞ value for DHA-dFdC was 677.3 μg/mL*h, ~363 μg/mL*h for the AUC0–48h. The plasma half-life of DHA-dFdC in the elimination phase was ~44 h. For a comparison, previously, we reported that when DHA-dFdC was given in a Tween 80-ethanol-water solution to mice at 75 mg/kg, its plasma half-life was only ~58 min (Naguib et al., 2016).

Figure 5.

Figure 5.

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Plasma DHA-dFdC concentration (μg/mL) at different time points (h) after DHA-dFdC-SLNs in suspension were intravenously injected into C57BL/6 mice. The dose of DHA-dFdC was 2 mg per mouse. Data (n = 3) were fitted using PKSolver, assuming a two-compartment model.

Table 2.

Selected plasma PK parameters of DHA-dFdC after DHA-dFdC-SLNs were given intravenously to mice.

Parameter Unit Observed
K10 1/h 0.02
K12 1/h 0.32
k21 1/h 0.58
t1/2α h 0.76
t1/2β h 43.95
C0 μg/mL 16.85
V mL 118.72
CL mL/h 2.95
V2 mL 66.66
CL2 mL/h 38.38
AUC0–48h (μg/mL)*h 362.82
AUC0-inf (μg/mL)*h 677.30
AUMC (μg/mL)*h2 42519.06
MRT h 62.78
Vss mL 185.38
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K10, rate constant of elimination; k12, rate constant of distribution; k21, rate constant of redistribution; t1/2α, initial or disposition half-life; t1/2β, terminal elimination half-life; C0, concentration at time t = 0; V, volume of distribution; CL, clearance; V2, volume of peripheral tissue compartment; CL2, inter-compartmental distribution; AUC0–48h, area under the curve (t = 0–48 h); AUC0-inf, area under the curve (t = 0-infinity); Vss, volume of distribution at steady state.

3.5. Evaluation of the antitumor activity of DHA-dFdC-SLNs in a mouse model

The antitumor activity of DHA-dFdC-SLNs was evaluated in mice with pre-established B16-F10 tumors. As shown in Fig. 6A, tumors grew aggressively when mice were left untreated or treated with the Tween 80-ethanol-in-water vehicle only. DHA-dFdC in solution and Blank-SLNs (DHA-dFdC-free SLNs) at the dosing regimen tested delayed the tumor growth by 4 days, but there was not any significant difference between the sizes of the tumors in mice treated with DHA-dFdC in solution or DHA-dFdC-free SLNs and the sizes of tumors in mice left untreated in all the days compared (Fig. 6A). DHA-dFdC-SLNs were most effective in inhibiting the tumor growth; the nanoparticle formulation delayed the tumor growth by close to 8 days, which is significant considering that mice bearing B16-F10 tumors, if left untreated, needed to be euthanized within 14 days after tumor cell injection due to aggressive tumor growth (Fig. 6A). Moreover, the sizes of the tumors in mice that were treated with the DHA-dFdC-SLNs were significantly smaller than those in mice that were left untreated or treated with the DHA-dFdC in solution (Fig. 6A). There was not any significant difference in the body weights of mice among the groups during the treatments (Fig. 6B), indicating DHA-dFdC-SLNs at the dosing regimen tested were well tolerated. Shown in Fig. 7 are representative H&E images of B16-F10 tumors from mice in different groups. Tumors in mice that were left untreated (Fig. 7A) or treated with vehicle (Fig. 7B) or DHA-dFdC-free SLNs (Fig. 7C) were in a late tumor stage with large blood vessels with large lumen. In addition, tumors in these groups showed large necrotic areas, increase in the desmoplasia, and vascular collapse (Fig. 7A–C). In solid tumors such as melanoma, high interstitial fluid constitutes a significant barrier to chemotherapy as it induces the compression of blood vessels, diverting the blood from the center of tumors to the periphery, which reduces the transcapillary transport of chemotherapeutics (Pautu et al., 2017). Tumor treated with DHA-dFdC-SLNs showed a higher number of blood vessels with small lumen (Fig. 7G). In addition, an increasing level of connective tissue can be observed around the tumoral zone in mice treated with DHA-dFdC-SLNs (Fig. 7F). In fact, this connective tissue may have tumor encapsulation effect, providing a protective barrier to tumor local and vascular invasion as previously suggested (Ng et al., 1992). For example, it was reported that patients with liver metastasis have a better prognostic when metastasis encapsulation occurs by the formation of a fibrotic capsule (Lunevicius et al., 2001; Morino et al., 1991; Ohlsson et al., 1998). It was suggested that the formation of capsules protects the liver parenchyma from cancer invasion (Lunevicius et al., 2001). This piece of evidence supports the use of DHA-dFdC-SLNs to treat melanoma, inducing the tumor encapsulation for further removal by surgery and avoiding metastasis due to the protective effect of this capsule. On the other hand, tumors in mice treated with DHA-dFdC showed vascular collapse and necrotic areas (Fig. 7D–E). Tumors in mice treated with DHA-dFdC-SLNs shown more cells in apoptosis, but less cells in necrosis, as compared to tumors in mice treated with DHA-dFdC in solution or left untreated. In this study, DHA-dFdC-free SLNs showed a tendency to delay tumor growth as compared to the untreated group (Fig. 6A). In fact, data from previous in vivo and in vitro studies showed that TPGS has anticancer activity as a single agent, being able to inhibit the growth of human prostate and lung carcinoma cells (Constantinou et al., 2012; Youk et al., 2005). In addition, it was reported that TPGS selectively induces apoptosis in T cell acute lymphocytic leukemia (ALL) or Jurkat clone E6–1 cells through the induction of oxidative stress pathway (Ruiz-Moreno et al., 2016). Moreover, TPGS was reported to selectively induce cell cycle arrest and apoptosis in breast cancer cell lines such as MCF7 and MDA-MB-231, but not in “normal” immortalized cells such as MCF-10A and MCF-12F (Neophytou et al., 2014). Finally, a synergistic effect between TPGS and docetaxel was reported in MCF-7 cell lines, wherein the incubation of MCF-7 cells with TPGS micelles without docetaxel induced cytotoxicity. One reason that could explain the lack of cytotoxicity by our DHA-dFdC-free SLNs against cells in culture is the low concentration of TPGS used in the formulation (< 1 μM). In previous studies, higher concentrations TPGS were used to induce cell cytotoxicity in culture (e.g. > 10 μM) or suppress tumor growth in animal studies (Constantinou et al., 2012; Neophytou et al., 2014; Ruiz-Moreno et al., 2016; Youk et al., 2005).

Figure 6.

Figure 6.

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Antitumor activity of DHA-dFdC-SLNs against B16-F10 tumor in a mouse model. (A) Tumor growth curve (▲, days at which treatment was given). (B) Mouse body weight change curves. C57BL/6 mice were s.c. injected with B16-F10 tumor cells on day 0. On day 7, mice were randomized into 5 groups (n = 5–6) and i.v. injected with DHA-dFdC-SLNs, DHA-dFdC in vehicle, or Blank-SLNs (DHA-dFdC-free SLNs) on days 7, 10, 13, and 16. The dose of DHA-dFdC was 50 mg/kg. As controls, one group of mice were left untreated. Data shown are mean ± SEM. p < 0.05; a DHA-dFdC-SLNs vs untreated; b DHA-dFdC-SLNs vs DHA-dFdC; c DHA-dFdC-SLNs vs Blank-SLNs; d DHA-dFdC-SLNs vs vehicle.

Figure 7.

Figure 7.

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Representative H&E images of B16-F10 tumors in C57BL/6 mice i.v. injected with DHA-dFdC-SLNs, DHA-dFdC-free SLNs, DHA-dFdC in vehicle, vehicle alone, or left untreated. Mice were euthanized on day 17 to collect tumor tissues. Tumor tissues of untreated (A), vehicle (B), and Blank-SLNs (C) groups are represented at a magnification 200X; while DHA-dFdC (D-E) and DHA-dFdC-SLNs (F-G) groups are represented by two different magnifications (100X, left; 200X, right). The scale bars in the 100 X images represent 100 μm, and that in the 200 X images represent 50 pm. Black circles represent tumor area, white dashed lines represent necrotic area, yellow arrows represent apoptotic cells, yellow asterisk represent desmoplasia, white arrows represent blood vessel, white crosses represent infiltration areas, white squares represent connective tissue areas, and yellow stars represent necrotic cells.

In summary, herein we report a new solid-lipid nanoparticle formulation of DHA-dFdC. All materials used in the formulation are biocompatible. Indeed, lecithin, GMS, and Tween 20 are all GRAS materials for parenteral administration (Rowe et al., 2009). TPGS is considered a safe pharmaceutical excipient that allows its use in parenteral pharmaceutical formulations (Rowe et al., 2009). Moreover, the method of preparing the DHA-dFdC-SLN formulation is straight forward and potentially scalable for industrial manufacturing. In addition, the small size of the DHA-dFdC-SLNs (~100 nm) allows their sterilization by filtration (0.2 μm). Finally, toxic organic solvents were not used when preparing the SLNs, avoiding evaporation process and residual solvent in the formulation. As to the mechanism underlying the improved antitumor activity of the DHA-dFdC-SLNs in the animal model, the enhance permeability and retention effect (EPR) in solid tumor tissues was likely applicable (Bazak et al., 2014).

4. Conclusion

Previously we synthesized DHA-dFdC and showed it has potent, broad-spectrum antitumor activity. Herein, we report the development of a new formulation of DHA-dFdC, DHA-dFdC-SLNs of 100 nm in diameter, that increased the apparent water solubility of DHA-dFdC by more than 200-fold and significantly increased its chemical stability. The DHA-dFdC-SLNs also increased the cytotoxicity of the DHA-dFdC in certain tumor cell lines, favorably modified the plasma pharmacokinetics of the DHA-dFdC, and enhanced its antitumor activity in a mouse model. Solid lipid nanoparticles, especially the ones contain antioxidant such as TPGS, represent a viable formulation for cancer chemotherapeutic agent/compounds that have poor water solubility and sensitive to oxidation. Future studies with DHA-dFdC-SLNs include safety as well as additional efficacy tests in animal models.

Acknowledgements

This work was supported in part by the National Cancer Institute of the National Institutes of Health (grant numbers CA179362 & CA135274) and the Alfred and Dorothy Mannino Fellowship in Pharmacy at UT Austin (to Z.C.). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health. S.A.V. was supported in part by the Becas-Chile Scholarship from the Government of Chile. R.F.A. was supported in part by a scholarship from the King Saud University. S.G.T. was supported in part by the University Graduate Continuing Fellowship from UT Austin.

List of abbreviations

DHA-dFdC

4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine

DHA

docosahexaenoic acid

DMSO

dimethyl sulfoxide

DMEM

Dulbecco’s Modified Eagle Media

dFdC

2′, 2′-difluorodeoxycytidine

FBS

fetal bovine serum

GPC

gel permeation chromatography

HPLC

High-performance liquid chromatography

i.v.

intravenous

Kcps

kilo counts per second

MTT

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

NMR

nuclear magnetic resonance

PEG

polyethylene glycol

PK

pharmacokinetic

PBS

phosphate-buffered saline

PUFA

polyunsaturated fatty acid

rcf

relative centrifugal force

SLNs

solid lipid nanoparticles

TEM

transmission electron microscopy

THF

tetrahydrofuran

TPGS

D-α-tocopherol polyethylene glycol 1000 succinate

GMS

glycerol monostearate

s.c.

subcutaneous

Footnotes

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