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Intratumoral gold-doxorubicin is effective in treating melanoma in mice
NANO-01108; No of Pages 11
Nanomedicine: Nanotechnology, Biology, and Medicine xx (2015) xxx – xxx nanomedjournal.com
Intratumoral gold-doxorubicin is effective in treating melanoma in mice Xuan Zhang, B.S. a , Jose G. Teodoro, PhD b, c , Jay L. Nadeau, PhD a,⁎
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Department of Biomedical Engineering, McGill University, Montreal QC Canada b Department of Biochemistry, McGill University, Montreal QC Canada c Goodman Cancer Research Centre, McGill University, Montreal QC Canada Received 20 November 2014; accepted 1 April 2015
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Abstract
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Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice. Au-Dox suppresses growth of B16 tumors in immunocompetent mice by N 70% for at least 19 days. In SK-MEL-28 xenografts, Au-Dox suppresses tumor growth almost completely for N 13 weeks, while tumors treated with Dox alone demonstrate accelerated growth after 10 weeks. Histological analysis showed significant apoptosis and necrosis in Au-Dox treated tumors. Intratumoral injection was significantly more effective than intravenous injection, which led to significant accumulation in liver and kidney with sub-therapeutic concentrations of Au-Dox. However, IV injection did not lead to significant damage in non-target organs, so improved targeting should permit this mode of delivery with little risk of systemic toxicity. The current construct is suitable for tumors accessible to intratumoral injection and represents a viable approach for solid tumors with native or acquired doxorubicin resistance. © 2015 Published by Elsevier Inc.
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Key words: Gold nanoparticles; Doxorubicin; Melanoma; B16; Apoptosis; Necrosis; in vivo; Intratumoral; Allograft; Xenograft
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Background
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Doxorubicin (Dox) is among the most widely used anticancer drugs for solid tumors. 1 It is a cytostatic anthracycline antibiotic with multiple modes of action, the most important of which are nuclear: Dox inhibits RNA synthesis by binding to RNA polymerase II and topoisomerase II 2 and intercalates into DNA, preventing replication. However, membrane-bound Dox may also damage cells by generation of reactive oxygen species (ROS). 3 Resistance is a major problem in Dox therapy 4; a few cancers, such as melanoma, are naturally Dox-resistant, whereas many others develop resistance after a round of treatment. Cardiotoxicity limits the cumulative dose that can be given. 5,6
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The authors declare no competing financial interests. JGT and JLN acknowledge the NSERC/CIHR CHRP Grant 385909-10; JLN acknowledges the MDEIE PSR-SIIRI-562 and NSERC Individual Discovery RGPIN 312970-2013; and JGT was supported by a grant from the CIHR (MOP-115195). EZ is supported by an FRSQ doctoral award, and JLN acknowledges salary support from Canada Research Chairs. ⁎Corresponding author at: Department of Biomedical Engineering, McGill University, Montreal QC Canada H3A 2B4. E-mail address: [email protected] (J.L. Nadeau).
Possible methods to improve tumor response and reduce toxicity include improved targeting of Dox to tumors, modification of the molecular structure to enhance the toxicity to cancer cells vs. healthy cells, or administration of beta-blockers to protect the heart during treatment. 7 Liposomal formulations of Dox are in clinical use. 8 Dendrimer formulations may improve pharmacokinetic performance over liposomes. 9,10 Although side effects still limit the dose that can be administered, cardiotoxicity is reduced compared to free Dox. 11 Numerous nanoparticle-Dox formulations with either Dox enclosed in the particle or attached to the surface are in early stages of development. Both organic and inorganic nanoparticles may be targeted to tumors actively or passively. 12 Active targeting involves conjugating an antibody, peptide, or receptor ligand to the nanoparticle that binds with a protein known to be overexpressed in the target cancer. It has not been established whether this approach is superior to passive targeting via the enhanced penetration and retention (EPR) effect, where the blood vessel leakiness and impaired lymphatic drainage of cancers causes accumulation of nanometer-sized particles. 13 The EPR effect has been well established in murine models, though not proven to occur in human cancers. 14 Although it is widely believed that long nanoparticle circulation times (days) are
http://dx.doi.org/10.1016/j.nano.2015.04.001 1549-9634/© 2015 Published by Elsevier Inc. Please cite this article as: Zhang X., et al., Intratumoral gold-doxorubicin is effective in treating melanoma in mice. Nanomedicine: NBM 2015;xx:1-11, http://dx.doi.org/10.1016/j.nano.2015.04.001
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Methods
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Unless otherwise specified, chemicals were purchased from Sigma-Aldrich Canada at the highest grade available and used as delivered.
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Au-Tiopronin and conjugation
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The procedure for gold nanoparticle synthesis was adapted from the literature 21; details are given in the Supplementary Material. Conjugation to doxorubicin was carried out by mixing nanoparticles, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in borate buffer (50 mM pH 8.8) for 1 h. Dox was then added to the mixture to a final concentration of 1 μM for Au, 50 μM for Dox, 5 mM for EDC, and 10 mM for NHS. Conjugation was performed in the dark at 20 °C for 24 h followed by filtration through a 3K MWCO membrane and washed 3x with dH2O (sterilized milli-Q water).
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Cell culture and toxicity
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Cells were cultured in high-glucose Dulbecco's minimum essential medium (DMEM, Invitrogen Canada, Burlington, ON) supplemented with L-glutamine (0.2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and fetal bovine serum (FBS, 10%), and incubated in a 5% CO2 atmosphere. The IC50 of Dox and Au-Dox was determined using the sulforhodamine B (SRB) assay. The SRB dye has a strong binding affinity to cellular proteins upon fixation with trichloroacetic acid. Therefore, the intensity of SRB dye confers the amount of protein, which is directly proportional to the number of cells. Cells were seeded at 5 x 10 3 cells per well in 96 well culture plates. When they had grown to 60% confluency, they were washed with PBS, incubated with AuNPs alone, Dox alone, or Au-Dox at various concentrations for 40 min in PBS, then washed with PBS and incubated in 200 μL of supplemented DMEM. After 24 h or 48 h, cells were fixed with trichloroacetic acid (60 μL of 40 % v/v) at 4 °C for 2 h, washed five times with distilled water, air-dried overnight, and stained with SRB reagent (50 μL) for 30 min. Unbound SRB was removed with 1% acetic acid, then bound SRB was dissolved in Tris (100 μL of 10 mM solution at pH 10.5). Absorbance was read at 500 nm. In each plate, at least 5 or 6 repeats of each condition were included, and independent assays were performed at least three times.
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Mechanisms of uptake of Au-Dox
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B16 cells were seeded in 6-well plates at 85,000 cells/well in supplemented DMEM. 24 h later, cells were treated with 10 μg/mL chlorpromazine, 2.5 mM methyl-β-cyclodextrin, or 100 μM
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With IV injection, a robust EPR effect was observed after one IV injection, but repeated injections over 2 weeks led to massive accumulation of Au in liver and kidney with sub-therapeutic levels in tumor. Injections of Dox alone at the same concentration killed two out of three mice. These results suggest that Au-Dox is a promising intratumoral agent that presents low risk of damage to non-target organs. For intravenous delivery, improved targeting of the particles will be necessary.
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required for the EPR effect, 15 a recent study demonstrated a robust EPR effect in a murine breast cancer model using 3.3 nm hydrodynamic diameter gold particles that were rapidly cleared by the kidney. 16 This study suggests that inorganic solid nanoparticles for cancer imaging and therapy should be designed to permit renal clearance. This avoids the potential long-term risk of metals or semiconductors in the liver and spleen and facilitates regulatory approval. Our approach is unique in two ways: by the use of ultrasmall particles conjugated to Dox, and by the use of a stable amide bond connecting the Dox to the particle, rather than a cleavable bond that releases free Dox. Cell killing by this stable Au-Dox conjugate occurs through binding of the intact conjugate to cellular structures, and therefore exhibits different mechanisms of action than conjugates from which Dox is released. Both of these aspects (particle size and stability) are essential for enhanced activity against Dox-resistant cells with simultaneous reduction in toxicity to Dox-sensitive cells and to non-target organs. We have conducted two previous in-depth studies of the behavior of this conjugate in vitro. In the first study 17 we found that that this conjugate was taken up by B16 melanoma cells more efficiently than Dox alone and approximately 6-fold faster. The IC50 of ultrasmall, stably conjugated Au-Dox was over 20-fold lower in B16 cells than that of Dox alone, but was nearly identical to that of Dox alone in cells that did not show Dox resistance. The mechanism of cell death in Au-Dox treated cells was mostly caspase-independent, with a minority of cells showing apoptosis. These results showed that the conjugate was able to overcome mechanisms of resistance in B16 cells, consistent with previous studies using transferrin-Dox conjugates, which showed that stably conjugated Dox can cause membrane damage to resistant cancer cells and necrosis, with less of an effect on nonmalignant cells. 18,19 In a second study, we used fluorescence lifetime imaging microscopy (FLIM) to track uptake and processing of Au-Dox in B16 cells. This showed strong association of intact Au-Dox conjugates with internal membrane structures, with slower but efficient penetration of bound Au-Dox into the nucleus. Cell structures were destroyed within 12-24 h, with a pattern consistent with necrosis rather than apoptosis. 20 In the current study, we compare the efficacy of ultrasmall, stably conjugated Au-Dox to Dox alone using two mouse models of melanoma. The first model used is B16, which is a murine cell line that creates aggressive tumors in immunocompetent mice. Intratumoral and intravenous (IV) delivery were investigated. Intratumoral injection was highly effective. Au-Dox produced N 70% reduction in tumor growth in B16 tumors that persisted for at least 19 days; Dox alone was able to suppress tumors for a shorter time before the growth accelerated. Due to their aggressiveness and large size, which included a large amount of necrotic material, B16 tumors were impossible to eliminate entirely. Histologic examination of sections showed uptake of Au into cells and nuclei. We also investigated xenografts of SK-MEL-28, a human melanoma cell line that can form tumors in nude mice. Au-Dox was able to completely suppress growth of SK-MEL-28 tumors (total duration of experiment N 6 months), whereas Dox alone showed early inhibition of tumors followed by rapid tumor growth.
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B16 and SK-MEL-28 tumor studies
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All animal experiments were conducted in accordance with the McGill Animal Ethics Committee guidelines. C57/BL6 mice and nude mice were purchased from Charles River Laboratories and Taconic Farm, respectively. There were 9 C57/BL6 mice at 10 weeks and 8 nude mice at 6 weeks in each treatment group. Tumors were established by inoculating subcutaneously 1 × 10 6 B16 cells or 5 × 10 6 SK-MEL-28 cells onto male C57/BL6 mice or female nude mice, respectively. The flank of C57/BL6 mice was shaved for implantation. C57/BL6 mice were given intratumoral injections (100 μL AuNPs alone, Dox alone, or Au-Dox at 100 μM of Dox or 4 μL of Au in PBS) three times a week starting at 9 days post implantation. The injection was carried out subcutaneously around the tumor if its size was below 500 mm 3 to avoid mechanical disruption. Nude mice received subcutaneous injections containing the same formulations twice a week starting 1 month post implantation due to the slow tumor development. The length, width, and height of tumors were measured daily with calipers. Experiments were terminated when tumor volumes in the control groups reached the clinical endpoint (2500 mm 3 ) in accordance with animal ethics guidelines. Tumor, kidney, and liver were collected for toxicity analysis. The tumor volume was calculated as: V ol ¼ Lwidth Llength Lheight ; and the tumor growth inhibition values (TGI) 2 were formula: T GI ¼ 100% calculated with the following V ol treated=final −V ol treated=initial 1− V ol control=final . −V ol control=initial
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B16 melanomas were established as above. 10 days later, AuNPs or Au-Dox at 1 μM of Au or 25 μM Dox (equivalent to 8.25 μg Au core, 16.5 μg Au-Tiopronin, and 3.4 μg Dox) in 250 μL PBS was injected intravenously in the tail vein. For the experiment involving 4 injections, injections were given twice a week. Organs were collected, weighed, washed with PBS, and flash frozen at –80 °C 1 h or 24 h post injection. Organs were then dissolved in 2 mL of fresh aqua regia (3:1 ratio of 37% HCl and 70% HNO3) at 40 °C until fully dissolved, and then the temperature was ramped up to 100 °C to evaporate the solution completely. The remnant was dissolved in 1 mL of 0.1% trace metal grade HCl followed by analysis by graphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer Analyst 800).
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Histology
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Tissue samples were fixed in 10% formalin for 24 h, followed by 48 h fixation in 70% ethanol and embedding in paraffin. Tissue slides were sectioned to 4 μm thick and stained. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. The tumor slides were scanned with an Aperio XT slide scanner and processed with Aperio ImageScope. Terminal deoxynucleotidyl transferase dUTP nick-end-labeling (TUNEL) staining was performed using the ApoAlert DNA Fragmentation Assay kit (Clontech), and imaged with Zeiss LSM 510 Meta Confocor2 Confocal Microscope. Silver staining of histological sections was performed by a method adapted from the literature. 22 Electron microscopy was performed at the UCSB MEIAF facility (see Supplemental Information for details).
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Results
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The Au nanoparticles had a mean ± SD diameter 2.7 ± 0.9 nm as measured by TEM (Figure 1, A). They were negatively charged (zeta potential − 42 mV). Particles were conjugated to Dox via an amide bond (Figure 1, B, C). The amount of bound
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Determination of Au in organs
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verapamil in serum-free medium for 1 h. Chlorpromazine is a clathrin-mediated endocytosis inhibitor that causes clathrin to be sequestered in late endosomes, and methyl-β-cyclodextrin (M βCD) removes cholesterol from the cell membrane, thus inhibiting formation of caveolae. Verapamil reduces the effect of multi-drug resistance by blocking P-glycoprotein. Au-Dox or Dox were added to the medium at 3 μM Dox concentration and incubated for 1 h. Cells were collected, washed three times with PBS, and resuspended in 1 mL PBS. The cell-associated florescence was detected using a FACSCalibur flowcytometer at 10,000 per cells. The data were analyzed using FlowJo.
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Figure 1. Characterization of gold nanoparticles and conjugates. (A) TEM image of gold nanoparticles. (B) Schematic of Au-tiopronin-Dox (structures not to scale). (C) Gold nanoparticle and Dox to scale.
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to just under 1 μM Dox (corresponding to ~ 40 nM gold nanoparticles). 17 The same effect was not seen in cell lines that were sensitive to Dox. Resistance to Dox, but not to Au-Dox, could be conferred by transfection of the anti-apoptotic protein Bcl-2. The gold nanoparticles alone were non-toxic to melanoma cell lines up to 80 μM. 17 Here we compared these growth curves with a human melanoma cell line that is not particularly Dox-resistant, SK-MEL-28. In SK-MEL-28 cells after 48 h, IC50 was reduced approximately 2-fold, from 5.9 Dox alone to 3.3 nM Au-Dox (Figure 2A). To examine mechanisms of uptake, the effect of various inhibitors on accumulation of Dox vs. Au-Dox in cells was studied (Figure 2B). As measured by flow cytometry, B16 cells were able to take up more Au-Dox (median fluorescence intensity [MFI]: 293) than Dox (MFI: 268) during the 1 h incubation time. When chlorpromazine was present, the uptake of AuDox was decreased, from an MFI value of 293 to 224. Although the MFI value for Dox dropped as well (from 268 to 230), the difference was not significant, indicating that clathrin-mediated endocytosis was involved in the uptake of Au-Dox but not Dox. When methyl-β-cyclodextrin was added, the uptake of Au-Dox decreased (MFI from 293 to 244) while an increase was observed in the Dox uptake (MFI from 268 to 330). As doxorubicin is a small hydrophobic molecule, the removal of cholesterol from the cell membrane would enable a faster and easier diffusion; this effect was less necessary for the more hydrophilic Au-Dox. Verapamil, a Ca 2 +-channel blocker, had no effect on the uptake of either Dox or Au-Dox (data not shown).
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Figure 2. (A) Survival vs. Log[Dox] concentration (in nM) for B16 and SK-MEL-28 melanoma cells. Error bars are standard deviations for 3 replicates in each of 3 different experiments. The lines are fits to the Hill equation. (B) Uptake of Au-Dox or Dox measured by flow cytometry in the presence of selected inhibitors (CHL = chlorpromazine; MBC = methyl-β-cyclodextrin). The signal is the Dox fluorescence; the upper panel represents cells treated with Au-Dox, and the lower panel shows cells treated with Dox only.
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Dox per particle was estimated from UV-Vis absorbance spectra as previously described 17 using a Spectra Max Plus plate reader (Molecular Devices, Novato, CA); we estimated 25 ± 5 Dox molecules per Au nanoparticle remaining after dialysis. The conjugates were stable for at least 24 h at pH 5 and pH 7. 20
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Cytotoxicity in culture
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We previously reported that conjugation to gold nanoparticles reduced the IC50 of Dox nearly 20-fold in B16 cells, from 17 μM
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Biodistribution and damage to non-target organs
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After a single injection of AuNPs or Au-Dox (total of 5.6 μg of Au) into tumor-bearing mice, the accumulation of Au in organs was measured. For unconjugated Au nanoparticles, total Au all measured organs was 112 ± 24 ng after 1 h and 229 ± 91 ng after 24 h. Of this accumulation, for gold nanoparticles alone, approximately 19% was in the tumor at 1 h. About 29% was found in kidney, 24% in liver, and the remainder distributed among other organs, primarily lung. After 24 h, 41% of the unconjugated AuNPs were found in tumor, 30% in liver, and 19% in kidney. For Au-Dox, there was a significantly greater total accumulation of Au after 1 h, 170 ± 71 ng, of which 27% was in tumor, 38% in kidney, and 19% in liver. After 24 h, the total accumulated Au did not differ significantly from the value with Au alone: 236 ± 60 ng. Of this amount, 31% was in tumor, 31% in liver, and 28% in kidney (Figure 3A). These values suggested that a robust EPR effect was possible with these small, non-PEGylated particles, in accordance with what has been observed in a previous study. 16 However, a series of 4 injections over a time course of 14 days led to accumulation of microgram amounts of Au in liver and kidney, with only a small minority of particles found in the tumor (Figure 3B). Two out of three of the mice treated with Dox alone died (data not shown; there were no deaths in the Au-Dox group). We thus concluded that intratumoral injection was necessary to achieve therapeutic results with these particles. Even with intratumoral injection, some particles will escape into the bloodstream, so it is important to investigate possible
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toxicity to non-target organs. This was assessed by IV injection of 79.2 mg of Au-tiopronin (corresponding to 29.6 mg of Au core) followed by histological examination of liver and kidneys after 24 h and 7 days. The kidneys were especially of interest following a report of renal toxicity from Au-tiopronin. 23 We examined 12 slides from each animal; there were no visible signs of toxicity in liver and kidneys in any of the animals.
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Efficacy of intratumoral injection: B16 tumors and SK-MEL28 xenografts B16 cells produced large, aggressive tumors in C57B6 mice. The tumors in the control groups, representing sham (PBS) injections and AuNPs alone, maintained rapid growth with continual increase in tumor volume over the 16 d of the experiment, when all of the control-treated group reached the clinical endpoint of 2500 mm 3 tumor volume. Up until Day 11 Dox alone and Au-Dox were indistinguishable, with both treatment groups showing a distinctly slower growth rate than the control groups. On Day 11, tumor volumes were 330 ± 50 mm 3 for PBS, 363 ± 85 mm 3 for Au alone, 187 ± 28 mm 3 for Au-Dox, and 315 ± 100 mm 3 for Dox alone (P b 0.05 for the two treatment groups vs. the two control groups; others non-significant). This represented 65% inhibition of tumor growth by Dox or Au-Dox. After Day 11, the rate of tumor growth remained slow in the Au-Dox group, but more accelerated in
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Figure 3. Delivery and efficacy. (A, B) Biodistribution of AuNPs and Au-Dox in mice with B16 melanoma xenograft tumors (n = 27). The Au levels were measured by graphite furnace atomic absorption spectroscopy (AAS) in ppb (ng/g) and the result was multiplied by the organ weight to give the total amount in ng. Error bars give the standard deviation. (A) Results after one injection. (B) Results after 4 injections over 14 days. (C, D) Efficacy of intratumoral injections in B16 melanoma xenografts and SK-MEL-28 xenografts. (C) B16 tumor volume vs. time for intratumoral injections of Dox alone, Au-Dox, PBS, or AuNPs alone. The injections were given every 2-3 days; each injection of Au-Dox or Dox alone contained 0.2 mg/kg of doxorubicin. The injections containing AuNPs alone contained the same amount of Au as in the Au-Dox case. The error bars represent standard deviations of 12 mice in each group. (D) SK-MEL-28 tumor volume over 100 days; clinical endpoints were not reached. The error bars represent standard deviations of 12 mice in each group.
a linear fashion in the Dox-only group. At Day 19, the endpoint of the experiment, there was a substantial difference between Au-Dox and Dox alone. Tumor volumes were 1630 ± 300 mm 3 for Dox alone and 800 ± 160 mm 3 for Au-Dox (P b .07). In the control groups where most mice had to be sacrificed on day 16, volumes were 2000 ± 250 mm 3 for PBS and 1750 ± 270 mm 3 for Au alone, or an ~73% inhibition by Au-Dox (Figure 3C). These results were confirmed using luciferase-expressing B16 tumors (see Supporting Information Figures S4, S5). Au-Dox was also tested against Dox alone in SK-MEL-28 xenografts in nude mice (Figure 3D). After the 5 th week of the experiment, the Dox alone group began to show an increase in tumor growth rate. The same was not seen with Au-Dox; in this group, tumor growth was almost completely suppressed (volume not significantly different from the starting point). After 100 days, the clinical endpoint had still not been reached, and the difference between Dox and Au-Dox was statistically significant (P b 0.03). 2 out of the total 8 mice in the Au-Dox treated mice showed complete tumor regression (volume taken as 0 for statistical purposes; these mice are still alive 8 months after the beginning of the experiment).
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Gross histology of B16 xenograft tumors showed very large tumors in the control groups (PBS sham injected and AuNPs only),
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with some necrotic areas resulting from the rapid tumor growth (Figure 4, A, E). Dox treatment alone resulted in significant decrease in tumor volume, with areas of necrosis (Figure 4, B, F). In Au-Dox treated tumors (Figure 4, D, H, L), significant tumor shrinkage was readily apparent, with most of the residual tissue being necrotic with occasional apoptotic cells (Figure 4L). Microscopic examination under higher magnification showed highly cellular, vascularized tumors, with well maintained integrity in the controls (Figure 4, I, J). In Dox treated samples, there was a marked delineation between unaffected tumor and areas of successful treatment (Figure 4, C, G). In Au-Dox treated tumors, residual areas of unaffected tumor were rarely seen (Figure 4, D), and the release of melanin as a result of melanosome breakage and cell death gave the tumor sections a brown color (Figure 4L). SK-MEL-28 tumors were smaller and less aggressive than the B16 tumors; a relatively large Au-Dox treated tumor is shown in Figure 5 because others were too small for visualization. After Dox treatment, only a small fraction of the tumor (~ 7%) responded and became necrotic (Figure 5, A, C), while Au-Dox caused necrosis of about 30% of the tumor volume in this particular example (Figure 5, B, D). Infiltrations of inflammatory cells (e.g. neutrophils and macrophages) into the necrotic regions in Dox and Au-Dox treated tumors were observed. In tumors treated with Dox alone, these regions were small and limited in volume (Figure 5E). In Au-Dox treated tumors, a large mixture
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Figure 4. Images of hematoxylin/eosin (H.E.) stained sections of B16 tumors at different magnifications after injection of (A, E, I) PBS, (E, F, J) unconjugated gold nanoparticles, (C, G, K) doxorubicin, and (D, H, L) Au-doxorubicin conjugates. The pink areas indicate necrosis, with individual apoptotic cells appearing black (arrow).
of apoptotic bodies and inflammatory cells was observed (Figure 5F), often occupying the bulk of the tumor volume.
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TUNEL staining
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TUNEL staining of histology sections showed positive “hot spots” in Dox and Au-Dox treated B16 (Figure 6, A-D) and SK-MEL-28 (Figure 6, E-H) tumors. The spots were more intense in the Au-Dox case and represented a greater fraction of the cells than with Dox alone in both types of tumors. Nonetheless, most of the cell death observed with Au-Dox was necrotic.
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Silver enhancement staining and STEM
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It was important to establish that Au-Dox injected into tumors was able to enter the tumor cells themselves, rather than being restricted to cells of the reticuloendothelial system. Because the particles used in this study were so small, they could not be identified by light microscopy without silver enhancement. With silver enhancement, they could be unambiguously distinguished from melanin granules only when present in cell nuclei. Spherical areas 1-2 mm in diameter were found only in Au and Au-Dox treated sections and not in controls or Dox treated sections (Figure 7A). Their presence in B16 nuclei confirmed that Au-Dox could be taken up by tumor cells in vivo, and STEM images were taken to confirm this. Figure 7, B-D show high-resolution STEM images of tumor sections.
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Figure 5. H.E. staining of SK-MEL-28 tumors treated with Dox (A, C, E) or Au-Dox (B, D, F) at low (A, B), medium (C, D) and high (E, F) magnification.
Discussion
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This study examined the in vivo efficacy of Au-Dox conjugates against two types of melanoma models: murine B16 (Dox-resistant) and human SK-MEL-28 (sensitive). We had previously 17 found that the in vitro efficacy of Au-Dox is greatly improved over Dox alone in Dox-resistant cell lines, but less so in Dox-sensitive cells. This result was supported here, where the B16 cells showed a 20-fold reduction in IC50 with Au-Dox vs. Dox alone. SK-MEL-28 cells only showed a 2-fold reduction, but this represented a significant improvement with Au-Dox over Dox alone. One explanation for the improved performance of Au-Dox lies in the uptake mechanisms, studied here by flow cytometry. The Au-Dox conjugates are taken up by at least two kinds of endocytosis (clathrin-mediated and caveolae), while the Dox alone penetrate the cells through simple diffusion — a well-recognized uptake mechanism for Dox. 24,25
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It is less efficient than energy-dependent uptake, and this explains why uptake of Au-Dox is more rapid and more thorough than uptake of Dox alone. In mice, Au-Dox was substantially superior to Dox alone in both types of tumor tested. Because of their large, aggressive nature, B16 tumors are rarely completely cured; however, Au-Dox led to a significant reduction in tumor size vs. control. Histological analysis demonstrated that most of the residual tumor in the Au-Dox treated animals was necrotic, and STEM showed that the tumor cells were able to take up the particles. Dox alone was significantly less effective than Au-Dox at promoting and maintaining tumor regression. Although effective at first, Dox treated tumors eventually began growing rapidly again, whereas those treated with Au-Dox showed long term growth inhibition. The best results were obtained with intratumoral injection. Intratumoral therapy is used in melanoma that presents as a large
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primary tumor. Delivery of various therapies has been tested in mouse models via this route: immunotherapy, gene therapy, and micro RNAs. 26–28 Intratumoral delivery of adenoviral gene therapy has shown to be effective in spontaneous canine melanoma. 29 In our study, we found that SK-MEL-28 tumors could be almost completely controlled for N 100 days using weekly injections of Au-Dox. By comparison, Dox alone led to accelerated tumor growth after 40–50 days. Because previous reports have indicated minimal systemic biodistribution of nanoparticles post intratumoral injection, 30–32 we did not measure biodistribution from intratumoral injection. Instead we chose to study any possible adverse effects of Au-Dox on nontarget organs by delivering particles intravenously, which leads to substantial accumulation in liver and kidney. Because we did not see significant pathology in these organs even after IV delivery, we are confident that intratumoral injection poses very little risk of liver and kidney damage.
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Figure 6. TUNEL staining of histology sections. The left panels show fluorescence signal only; the right panels show overlay of fluorescence and DIC. (A) Low magnification image of hot spot in Dox-only treated B16 tumor. (B) High magnification image of hot spot in Dox treated B16 tumor. (C) Low magnification image of hot spot in Au-Dox treated B16 tumor. (D) High magnification image of hot spot in Au-Dox treated B16 tumor. (E) Low magnification image of hot spot in Dox-only treated SK-MEL-28 tumor. (F) High magnification image of hot spot in Dox-treated SK-MEL-28 tumor. (G) Low magnification image of hot spot in Au-Dox treated SK-MEL-28 tumor. (H) High magnification image of hot spot in Au-Dox treated SK-MEL-28 tumor.
Although some hot spots of apoptosis were seen with both Dox and Au-Dox, the primary mechanism of cell death in Au-Dox treated tumors was necrosis. Necrotic cell death is associated with physical insults, failure of osmotic regulation, and ATP depletion, 33,34 all of which are likely to be caused by massive uptake of Au-Dox through energy-dependent mechanisms. Necrosis also leads to inflammatory responses. Consistent with such effects, we observed extensive infiltration of immune cells throughout the SK-MEL-28 xenografts. The role of autophagy was not examined here, although it is a common mechanism of cell death associated with nanoparticle overload. 35–38 Future work in our laboratory will examine the mechanisms of cell death more closely in vitro and in vivo. Two factors limited the use of intravenous delivery in this study. The first was the toxicity of Dox alone, which led to death in 2/3 mice in a pilot study, making it difficult to perform appropriate control experiments. The second factor was the
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limited EPR effect seen after repeated Au-Dox and AuNP injections. After a single injection, a significant fraction of the nanoparticles were found in the tumor (close to 40%); however, with multiple injections over two weeks, microgram amounts of gold were seen in liver and kidney, with sub-therapeutic levels in tumor. Improving the targeting and excretion of particles will be necessary to make Au-Dox a useful agent for IV injection. Improved targeting may be possible by conjugating tumorpenetrating or receptor-binding peptides to the surface of the nanoparticles along with the Dox. One option for receptor targeting in melanoma is the melanocortin receptor, targeted by the tridecapeptide α-MSH (melanocyte stimulating hormone). MSH is produced by the brain and pituitary gland and is the most potent melanotropic peptide in the regulation of skin pigmentation via the MC1R. Radiolabeled α-MSH and its analogs have been used to image melanoma with essentially no uptake in non-target tissues except kidney. 39 Future studies will determine whether specific targeting improves uptake after IV
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Figure 7. Light and EM microscopy of histology sections from B16 tumors after IV injection of Au-Dox. (A) Silver staining. The black arrows indicate the Au. The white arrowhead shows nonspecific dark areas that are found in controls. The inset shows a high-magnification image of a single cell. (B) Low-magnification of STEM section, showing tumor cells, adipocytes (arrowhead), and deposits of Au (arrow). (C) Higher magnification image of B16 cell, showing intracellular and intranuclear Au. (D) STEM image showing Au within a capillary (arrows). (E,F) Cell nuclei in tumors with (E) and without (F) Au-Dox to illustrate the difference in contrast.
injection and whether it leads to greater efficacy after either IV or intratumoral injection. This study represents a promising step towards the development of clinical agents for tumors with native or acquired doxorubicin resistance. The synthesis procedure is simple, easily scaled to large volumes, and does not involve toxic precursors such as cetyltrimethylammonium bromide (CTAB). Tiopronin is FDA-approved for the prevention of kidney stones (trade name Thiola). The biggest hurdle will be producing the gold nanoparticles through a reproducible method and obtaining approval for their use as a drug, though there is significant precedent in this area. Several gold-chemotherapy agents are in clinical trials or seeking FDA approval. CytImmune’s goldtumor necrosis factor (TNF) conjugate, Aurimune, entered Phase I trials in 2006. 40 In the Phase I trial, no toxicity due to the gold was observed in the 16 human subjects. In fact, a reduction in toxicity was seen relative to free TNF, with the typical dose-limiting hypotension not seen with the conjugates. The
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drug is currently in Phase II trials for pancreatic cancer, melanoma, soft tissue sarcoma, ovarian, and breast cancer. Another gold preparation is Nanospectra’s “AuroLase” particles, used for photothermal therapy. 41–43 These particles have been approved for investigational use in refractory head and neck cancer and lung cancer. The greatest barrier to FDA approval of gold nanoparticle preparations for imaging is the slow rate of clearance of larger (10-15 nm) particles. 44 However, smaller particles clear more quickly, and the ultrasmall gold particles we report here were cleared by the kidneys in mice within several hours. The design principles presented here are also generalizable to other chemotherapeutic agents besides doxorubicin. Many traditional chemotherapeutic agents may be more effective when conjugated to nanoparticles, especially in synergy with receptor blockade and/or radiotherapy. 45,46 Progress in this area would open the door to a variety of potential treatment strategies for drug-resistant tumors.
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38. Hackenberg S, et al. Nanoparticle-induced photocatalytic head and neck squamous cell carcinoma cell death is associated with autophagy. Nanomedicine 2014;9:21-33. 39. McQuade P, et al. Imaging of melanoma using 64Cu- and 86Y-DOTAReCCMSH(Arg11), a cyclized peptide analogue of alpha-MSH. J Med Chem 2005;48:2985-92. 40. Goel R, Shah N, Visaria R, Paciotti GF, Bischof JC. Biodistribution of TNF-alpha-coated gold nanoparticles in an in vivo model system. Nanomedicine (Lond) 2009;4:401-10. 41. Stern JM, Stanfield J, Kabbani W, Hsieh JT, Cadeddu JA. Selective prostate cancer thermal ablation with laser activated gold nanoshells. J Urol 2008;179:748-53.
42. Morton JG, Day ES, Halas NJ, West JL. Nanoshells for photothermal cancer therapy. Methods Mol Biol 2010;624:101-17. 43. Day ES, et al. Vascular-targeted photothermal therapy of an orthotopic murine glioma model. Nanomedicine (Lond) 2012;7:1133-48. 44. Hall JB, Dobrovolskaia MA, Patri AK, McNeil SE. Characterization of nanoparticles for therapeutics. Nanomedicine (Lond) 2007;2:789-803. 45. Starkey JR, et al. New two-photon activated photodynamic therapy sensitizers induce xenograft tumor regressions after near-IR laser treatment through the body of the host mouse. Clin Cancer Res 2008;14:6564-73. 46. Atkinson RL, et al. Thermal enhancement with optically activated gold nanoshells sensitizes breast cancer stem cells to radiation therapy. Sci Transl Med 2010;2:55ra79.
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Graphical Abstract
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Intratumoral gold-doxorubicin is effective in treating melanoma in mice
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Xuan Zhang, B.S. a, Jose G. Teodoro, PhD b,c, Jay L. Nadeau, PhD a,⁎
Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2015) xxx – xxx
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Department of Biomedical Engineering, McGill University, Montreal QC Canada Department of Biochemistry, McGill University, Montreal QC Canada Goodman Cancer Research Centre, McGill University, Montreal QC Canada
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Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice.
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Intratumoral gold-doxorubicin is effective in treating melanoma in mice Xuan Zhang, B.S. a , Jose G. Teodoro, PhD b, c , Jay L. Nadeau, PhD a,⁎
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Department of Biomedical Engineering, McGill University, Montreal QC Canada b Department of Biochemistry, McGill University, Montreal QC Canada c Goodman Cancer Research Centre, McGill University, Montreal QC Canada Received 20 November 2014; accepted 1 April 2015
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Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice. Au-Dox suppresses growth of B16 tumors in immunocompetent mice by N 70% for at least 19 days. In SK-MEL-28 xenografts, Au-Dox suppresses tumor growth almost completely for N 13 weeks, while tumors treated with Dox alone demonstrate accelerated growth after 10 weeks. Histological analysis showed significant apoptosis and necrosis in Au-Dox treated tumors. Intratumoral injection was significantly more effective than intravenous injection, which led to significant accumulation in liver and kidney with sub-therapeutic concentrations of Au-Dox. However, IV injection did not lead to significant damage in non-target organs, so improved targeting should permit this mode of delivery with little risk of systemic toxicity. The current construct is suitable for tumors accessible to intratumoral injection and represents a viable approach for solid tumors with native or acquired doxorubicin resistance. © 2015 Published by Elsevier Inc.
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Key words: Gold nanoparticles; Doxorubicin; Melanoma; B16; Apoptosis; Necrosis; in vivo; Intratumoral; Allograft; Xenograft
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Background
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Doxorubicin (Dox) is among the most widely used anticancer drugs for solid tumors. 1 It is a cytostatic anthracycline antibiotic with multiple modes of action, the most important of which are nuclear: Dox inhibits RNA synthesis by binding to RNA polymerase II and topoisomerase II 2 and intercalates into DNA, preventing replication. However, membrane-bound Dox may also damage cells by generation of reactive oxygen species (ROS). 3 Resistance is a major problem in Dox therapy 4; a few cancers, such as melanoma, are naturally Dox-resistant, whereas many others develop resistance after a round of treatment. Cardiotoxicity limits the cumulative dose that can be given. 5,6
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The authors declare no competing financial interests. JGT and JLN acknowledge the NSERC/CIHR CHRP Grant 385909-10; JLN acknowledges the MDEIE PSR-SIIRI-562 and NSERC Individual Discovery RGPIN 312970-2013; and JGT was supported by a grant from the CIHR (MOP-115195). EZ is supported by an FRSQ doctoral award, and JLN acknowledges salary support from Canada Research Chairs. ⁎Corresponding author at: Department of Biomedical Engineering, McGill University, Montreal QC Canada H3A 2B4. E-mail address: [email protected] (J.L. Nadeau).
Possible methods to improve tumor response and reduce toxicity include improved targeting of Dox to tumors, modification of the molecular structure to enhance the toxicity to cancer cells vs. healthy cells, or administration of beta-blockers to protect the heart during treatment. 7 Liposomal formulations of Dox are in clinical use. 8 Dendrimer formulations may improve pharmacokinetic performance over liposomes. 9,10 Although side effects still limit the dose that can be administered, cardiotoxicity is reduced compared to free Dox. 11 Numerous nanoparticle-Dox formulations with either Dox enclosed in the particle or attached to the surface are in early stages of development. Both organic and inorganic nanoparticles may be targeted to tumors actively or passively. 12 Active targeting involves conjugating an antibody, peptide, or receptor ligand to the nanoparticle that binds with a protein known to be overexpressed in the target cancer. It has not been established whether this approach is superior to passive targeting via the enhanced penetration and retention (EPR) effect, where the blood vessel leakiness and impaired lymphatic drainage of cancers causes accumulation of nanometer-sized particles. 13 The EPR effect has been well established in murine models, though not proven to occur in human cancers. 14 Although it is widely believed that long nanoparticle circulation times (days) are
http://dx.doi.org/10.1016/j.nano.2015.04.001 1549-9634/© 2015 Published by Elsevier Inc. Please cite this article as: Zhang X., et al., Intratumoral gold-doxorubicin is effective in treating melanoma in mice. Nanomedicine: NBM 2015;xx:1-11, http://dx.doi.org/10.1016/j.nano.2015.04.001
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Methods
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Unless otherwise specified, chemicals were purchased from Sigma-Aldrich Canada at the highest grade available and used as delivered.
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Au-Tiopronin and conjugation
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The procedure for gold nanoparticle synthesis was adapted from the literature 21; details are given in the Supplementary Material. Conjugation to doxorubicin was carried out by mixing nanoparticles, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in borate buffer (50 mM pH 8.8) for 1 h. Dox was then added to the mixture to a final concentration of 1 μM for Au, 50 μM for Dox, 5 mM for EDC, and 10 mM for NHS. Conjugation was performed in the dark at 20 °C for 24 h followed by filtration through a 3K MWCO membrane and washed 3x with dH2O (sterilized milli-Q water).
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Cells were cultured in high-glucose Dulbecco's minimum essential medium (DMEM, Invitrogen Canada, Burlington, ON) supplemented with L-glutamine (0.2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and fetal bovine serum (FBS, 10%), and incubated in a 5% CO2 atmosphere. The IC50 of Dox and Au-Dox was determined using the sulforhodamine B (SRB) assay. The SRB dye has a strong binding affinity to cellular proteins upon fixation with trichloroacetic acid. Therefore, the intensity of SRB dye confers the amount of protein, which is directly proportional to the number of cells. Cells were seeded at 5 x 10 3 cells per well in 96 well culture plates. When they had grown to 60% confluency, they were washed with PBS, incubated with AuNPs alone, Dox alone, or Au-Dox at various concentrations for 40 min in PBS, then washed with PBS and incubated in 200 μL of supplemented DMEM. After 24 h or 48 h, cells were fixed with trichloroacetic acid (60 μL of 40 % v/v) at 4 °C for 2 h, washed five times with distilled water, air-dried overnight, and stained with SRB reagent (50 μL) for 30 min. Unbound SRB was removed with 1% acetic acid, then bound SRB was dissolved in Tris (100 μL of 10 mM solution at pH 10.5). Absorbance was read at 500 nm. In each plate, at least 5 or 6 repeats of each condition were included, and independent assays were performed at least three times.
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Mechanisms of uptake of Au-Dox
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B16 cells were seeded in 6-well plates at 85,000 cells/well in supplemented DMEM. 24 h later, cells were treated with 10 μg/mL chlorpromazine, 2.5 mM methyl-β-cyclodextrin, or 100 μM
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With IV injection, a robust EPR effect was observed after one IV injection, but repeated injections over 2 weeks led to massive accumulation of Au in liver and kidney with sub-therapeutic levels in tumor. Injections of Dox alone at the same concentration killed two out of three mice. These results suggest that Au-Dox is a promising intratumoral agent that presents low risk of damage to non-target organs. For intravenous delivery, improved targeting of the particles will be necessary.
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required for the EPR effect, 15 a recent study demonstrated a robust EPR effect in a murine breast cancer model using 3.3 nm hydrodynamic diameter gold particles that were rapidly cleared by the kidney. 16 This study suggests that inorganic solid nanoparticles for cancer imaging and therapy should be designed to permit renal clearance. This avoids the potential long-term risk of metals or semiconductors in the liver and spleen and facilitates regulatory approval. Our approach is unique in two ways: by the use of ultrasmall particles conjugated to Dox, and by the use of a stable amide bond connecting the Dox to the particle, rather than a cleavable bond that releases free Dox. Cell killing by this stable Au-Dox conjugate occurs through binding of the intact conjugate to cellular structures, and therefore exhibits different mechanisms of action than conjugates from which Dox is released. Both of these aspects (particle size and stability) are essential for enhanced activity against Dox-resistant cells with simultaneous reduction in toxicity to Dox-sensitive cells and to non-target organs. We have conducted two previous in-depth studies of the behavior of this conjugate in vitro. In the first study 17 we found that that this conjugate was taken up by B16 melanoma cells more efficiently than Dox alone and approximately 6-fold faster. The IC50 of ultrasmall, stably conjugated Au-Dox was over 20-fold lower in B16 cells than that of Dox alone, but was nearly identical to that of Dox alone in cells that did not show Dox resistance. The mechanism of cell death in Au-Dox treated cells was mostly caspase-independent, with a minority of cells showing apoptosis. These results showed that the conjugate was able to overcome mechanisms of resistance in B16 cells, consistent with previous studies using transferrin-Dox conjugates, which showed that stably conjugated Dox can cause membrane damage to resistant cancer cells and necrosis, with less of an effect on nonmalignant cells. 18,19 In a second study, we used fluorescence lifetime imaging microscopy (FLIM) to track uptake and processing of Au-Dox in B16 cells. This showed strong association of intact Au-Dox conjugates with internal membrane structures, with slower but efficient penetration of bound Au-Dox into the nucleus. Cell structures were destroyed within 12-24 h, with a pattern consistent with necrosis rather than apoptosis. 20 In the current study, we compare the efficacy of ultrasmall, stably conjugated Au-Dox to Dox alone using two mouse models of melanoma. The first model used is B16, which is a murine cell line that creates aggressive tumors in immunocompetent mice. Intratumoral and intravenous (IV) delivery were investigated. Intratumoral injection was highly effective. Au-Dox produced N 70% reduction in tumor growth in B16 tumors that persisted for at least 19 days; Dox alone was able to suppress tumors for a shorter time before the growth accelerated. Due to their aggressiveness and large size, which included a large amount of necrotic material, B16 tumors were impossible to eliminate entirely. Histologic examination of sections showed uptake of Au into cells and nuclei. We also investigated xenografts of SK-MEL-28, a human melanoma cell line that can form tumors in nude mice. Au-Dox was able to completely suppress growth of SK-MEL-28 tumors (total duration of experiment N 6 months), whereas Dox alone showed early inhibition of tumors followed by rapid tumor growth.
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All animal experiments were conducted in accordance with the McGill Animal Ethics Committee guidelines. C57/BL6 mice and nude mice were purchased from Charles River Laboratories and Taconic Farm, respectively. There were 9 C57/BL6 mice at 10 weeks and 8 nude mice at 6 weeks in each treatment group. Tumors were established by inoculating subcutaneously 1 × 10 6 B16 cells or 5 × 10 6 SK-MEL-28 cells onto male C57/BL6 mice or female nude mice, respectively. The flank of C57/BL6 mice was shaved for implantation. C57/BL6 mice were given intratumoral injections (100 μL AuNPs alone, Dox alone, or Au-Dox at 100 μM of Dox or 4 μL of Au in PBS) three times a week starting at 9 days post implantation. The injection was carried out subcutaneously around the tumor if its size was below 500 mm 3 to avoid mechanical disruption. Nude mice received subcutaneous injections containing the same formulations twice a week starting 1 month post implantation due to the slow tumor development. The length, width, and height of tumors were measured daily with calipers. Experiments were terminated when tumor volumes in the control groups reached the clinical endpoint (2500 mm 3 ) in accordance with animal ethics guidelines. Tumor, kidney, and liver were collected for toxicity analysis. The tumor volume was calculated as: V ol ¼ Lwidth Llength Lheight ; and the tumor growth inhibition values (TGI) 2 were formula: T GI ¼ 100% calculated with the following V ol treated=final −V ol treated=initial 1− V ol control=final . −V ol control=initial
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B16 melanomas were established as above. 10 days later, AuNPs or Au-Dox at 1 μM of Au or 25 μM Dox (equivalent to 8.25 μg Au core, 16.5 μg Au-Tiopronin, and 3.4 μg Dox) in 250 μL PBS was injected intravenously in the tail vein. For the experiment involving 4 injections, injections were given twice a week. Organs were collected, weighed, washed with PBS, and flash frozen at –80 °C 1 h or 24 h post injection. Organs were then dissolved in 2 mL of fresh aqua regia (3:1 ratio of 37% HCl and 70% HNO3) at 40 °C until fully dissolved, and then the temperature was ramped up to 100 °C to evaporate the solution completely. The remnant was dissolved in 1 mL of 0.1% trace metal grade HCl followed by analysis by graphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer Analyst 800).
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Tissue samples were fixed in 10% formalin for 24 h, followed by 48 h fixation in 70% ethanol and embedding in paraffin. Tissue slides were sectioned to 4 μm thick and stained. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. The tumor slides were scanned with an Aperio XT slide scanner and processed with Aperio ImageScope. Terminal deoxynucleotidyl transferase dUTP nick-end-labeling (TUNEL) staining was performed using the ApoAlert DNA Fragmentation Assay kit (Clontech), and imaged with Zeiss LSM 510 Meta Confocor2 Confocal Microscope. Silver staining of histological sections was performed by a method adapted from the literature. 22 Electron microscopy was performed at the UCSB MEIAF facility (see Supplemental Information for details).
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The Au nanoparticles had a mean ± SD diameter 2.7 ± 0.9 nm as measured by TEM (Figure 1, A). They were negatively charged (zeta potential − 42 mV). Particles were conjugated to Dox via an amide bond (Figure 1, B, C). The amount of bound
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verapamil in serum-free medium for 1 h. Chlorpromazine is a clathrin-mediated endocytosis inhibitor that causes clathrin to be sequestered in late endosomes, and methyl-β-cyclodextrin (M βCD) removes cholesterol from the cell membrane, thus inhibiting formation of caveolae. Verapamil reduces the effect of multi-drug resistance by blocking P-glycoprotein. Au-Dox or Dox were added to the medium at 3 μM Dox concentration and incubated for 1 h. Cells were collected, washed three times with PBS, and resuspended in 1 mL PBS. The cell-associated florescence was detected using a FACSCalibur flowcytometer at 10,000 per cells. The data were analyzed using FlowJo.
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Figure 1. Characterization of gold nanoparticles and conjugates. (A) TEM image of gold nanoparticles. (B) Schematic of Au-tiopronin-Dox (structures not to scale). (C) Gold nanoparticle and Dox to scale.
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to just under 1 μM Dox (corresponding to ~ 40 nM gold nanoparticles). 17 The same effect was not seen in cell lines that were sensitive to Dox. Resistance to Dox, but not to Au-Dox, could be conferred by transfection of the anti-apoptotic protein Bcl-2. The gold nanoparticles alone were non-toxic to melanoma cell lines up to 80 μM. 17 Here we compared these growth curves with a human melanoma cell line that is not particularly Dox-resistant, SK-MEL-28. In SK-MEL-28 cells after 48 h, IC50 was reduced approximately 2-fold, from 5.9 Dox alone to 3.3 nM Au-Dox (Figure 2A). To examine mechanisms of uptake, the effect of various inhibitors on accumulation of Dox vs. Au-Dox in cells was studied (Figure 2B). As measured by flow cytometry, B16 cells were able to take up more Au-Dox (median fluorescence intensity [MFI]: 293) than Dox (MFI: 268) during the 1 h incubation time. When chlorpromazine was present, the uptake of AuDox was decreased, from an MFI value of 293 to 224. Although the MFI value for Dox dropped as well (from 268 to 230), the difference was not significant, indicating that clathrin-mediated endocytosis was involved in the uptake of Au-Dox but not Dox. When methyl-β-cyclodextrin was added, the uptake of Au-Dox decreased (MFI from 293 to 244) while an increase was observed in the Dox uptake (MFI from 268 to 330). As doxorubicin is a small hydrophobic molecule, the removal of cholesterol from the cell membrane would enable a faster and easier diffusion; this effect was less necessary for the more hydrophilic Au-Dox. Verapamil, a Ca 2 +-channel blocker, had no effect on the uptake of either Dox or Au-Dox (data not shown).
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Figure 2. (A) Survival vs. Log[Dox] concentration (in nM) for B16 and SK-MEL-28 melanoma cells. Error bars are standard deviations for 3 replicates in each of 3 different experiments. The lines are fits to the Hill equation. (B) Uptake of Au-Dox or Dox measured by flow cytometry in the presence of selected inhibitors (CHL = chlorpromazine; MBC = methyl-β-cyclodextrin). The signal is the Dox fluorescence; the upper panel represents cells treated with Au-Dox, and the lower panel shows cells treated with Dox only.
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Dox per particle was estimated from UV-Vis absorbance spectra as previously described 17 using a Spectra Max Plus plate reader (Molecular Devices, Novato, CA); we estimated 25 ± 5 Dox molecules per Au nanoparticle remaining after dialysis. The conjugates were stable for at least 24 h at pH 5 and pH 7. 20
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After a single injection of AuNPs or Au-Dox (total of 5.6 μg of Au) into tumor-bearing mice, the accumulation of Au in organs was measured. For unconjugated Au nanoparticles, total Au all measured organs was 112 ± 24 ng after 1 h and 229 ± 91 ng after 24 h. Of this accumulation, for gold nanoparticles alone, approximately 19% was in the tumor at 1 h. About 29% was found in kidney, 24% in liver, and the remainder distributed among other organs, primarily lung. After 24 h, 41% of the unconjugated AuNPs were found in tumor, 30% in liver, and 19% in kidney. For Au-Dox, there was a significantly greater total accumulation of Au after 1 h, 170 ± 71 ng, of which 27% was in tumor, 38% in kidney, and 19% in liver. After 24 h, the total accumulated Au did not differ significantly from the value with Au alone: 236 ± 60 ng. Of this amount, 31% was in tumor, 31% in liver, and 28% in kidney (Figure 3A). These values suggested that a robust EPR effect was possible with these small, non-PEGylated particles, in accordance with what has been observed in a previous study. 16 However, a series of 4 injections over a time course of 14 days led to accumulation of microgram amounts of Au in liver and kidney, with only a small minority of particles found in the tumor (Figure 3B). Two out of three of the mice treated with Dox alone died (data not shown; there were no deaths in the Au-Dox group). We thus concluded that intratumoral injection was necessary to achieve therapeutic results with these particles. Even with intratumoral injection, some particles will escape into the bloodstream, so it is important to investigate possible
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toxicity to non-target organs. This was assessed by IV injection of 79.2 mg of Au-tiopronin (corresponding to 29.6 mg of Au core) followed by histological examination of liver and kidneys after 24 h and 7 days. The kidneys were especially of interest following a report of renal toxicity from Au-tiopronin. 23 We examined 12 slides from each animal; there were no visible signs of toxicity in liver and kidneys in any of the animals.
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Efficacy of intratumoral injection: B16 tumors and SK-MEL28 xenografts B16 cells produced large, aggressive tumors in C57B6 mice. The tumors in the control groups, representing sham (PBS) injections and AuNPs alone, maintained rapid growth with continual increase in tumor volume over the 16 d of the experiment, when all of the control-treated group reached the clinical endpoint of 2500 mm 3 tumor volume. Up until Day 11 Dox alone and Au-Dox were indistinguishable, with both treatment groups showing a distinctly slower growth rate than the control groups. On Day 11, tumor volumes were 330 ± 50 mm 3 for PBS, 363 ± 85 mm 3 for Au alone, 187 ± 28 mm 3 for Au-Dox, and 315 ± 100 mm 3 for Dox alone (P b 0.05 for the two treatment groups vs. the two control groups; others non-significant). This represented 65% inhibition of tumor growth by Dox or Au-Dox. After Day 11, the rate of tumor growth remained slow in the Au-Dox group, but more accelerated in
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Figure 3. Delivery and efficacy. (A, B) Biodistribution of AuNPs and Au-Dox in mice with B16 melanoma xenograft tumors (n = 27). The Au levels were measured by graphite furnace atomic absorption spectroscopy (AAS) in ppb (ng/g) and the result was multiplied by the organ weight to give the total amount in ng. Error bars give the standard deviation. (A) Results after one injection. (B) Results after 4 injections over 14 days. (C, D) Efficacy of intratumoral injections in B16 melanoma xenografts and SK-MEL-28 xenografts. (C) B16 tumor volume vs. time for intratumoral injections of Dox alone, Au-Dox, PBS, or AuNPs alone. The injections were given every 2-3 days; each injection of Au-Dox or Dox alone contained 0.2 mg/kg of doxorubicin. The injections containing AuNPs alone contained the same amount of Au as in the Au-Dox case. The error bars represent standard deviations of 12 mice in each group. (D) SK-MEL-28 tumor volume over 100 days; clinical endpoints were not reached. The error bars represent standard deviations of 12 mice in each group.
a linear fashion in the Dox-only group. At Day 19, the endpoint of the experiment, there was a substantial difference between Au-Dox and Dox alone. Tumor volumes were 1630 ± 300 mm 3 for Dox alone and 800 ± 160 mm 3 for Au-Dox (P b .07). In the control groups where most mice had to be sacrificed on day 16, volumes were 2000 ± 250 mm 3 for PBS and 1750 ± 270 mm 3 for Au alone, or an ~73% inhibition by Au-Dox (Figure 3C). These results were confirmed using luciferase-expressing B16 tumors (see Supporting Information Figures S4, S5). Au-Dox was also tested against Dox alone in SK-MEL-28 xenografts in nude mice (Figure 3D). After the 5 th week of the experiment, the Dox alone group began to show an increase in tumor growth rate. The same was not seen with Au-Dox; in this group, tumor growth was almost completely suppressed (volume not significantly different from the starting point). After 100 days, the clinical endpoint had still not been reached, and the difference between Dox and Au-Dox was statistically significant (P b 0.03). 2 out of the total 8 mice in the Au-Dox treated mice showed complete tumor regression (volume taken as 0 for statistical purposes; these mice are still alive 8 months after the beginning of the experiment).
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Gross histology of B16 xenograft tumors showed very large tumors in the control groups (PBS sham injected and AuNPs only),
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with some necrotic areas resulting from the rapid tumor growth (Figure 4, A, E). Dox treatment alone resulted in significant decrease in tumor volume, with areas of necrosis (Figure 4, B, F). In Au-Dox treated tumors (Figure 4, D, H, L), significant tumor shrinkage was readily apparent, with most of the residual tissue being necrotic with occasional apoptotic cells (Figure 4L). Microscopic examination under higher magnification showed highly cellular, vascularized tumors, with well maintained integrity in the controls (Figure 4, I, J). In Dox treated samples, there was a marked delineation between unaffected tumor and areas of successful treatment (Figure 4, C, G). In Au-Dox treated tumors, residual areas of unaffected tumor were rarely seen (Figure 4, D), and the release of melanin as a result of melanosome breakage and cell death gave the tumor sections a brown color (Figure 4L). SK-MEL-28 tumors were smaller and less aggressive than the B16 tumors; a relatively large Au-Dox treated tumor is shown in Figure 5 because others were too small for visualization. After Dox treatment, only a small fraction of the tumor (~ 7%) responded and became necrotic (Figure 5, A, C), while Au-Dox caused necrosis of about 30% of the tumor volume in this particular example (Figure 5, B, D). Infiltrations of inflammatory cells (e.g. neutrophils and macrophages) into the necrotic regions in Dox and Au-Dox treated tumors were observed. In tumors treated with Dox alone, these regions were small and limited in volume (Figure 5E). In Au-Dox treated tumors, a large mixture
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Figure 4. Images of hematoxylin/eosin (H.E.) stained sections of B16 tumors at different magnifications after injection of (A, E, I) PBS, (E, F, J) unconjugated gold nanoparticles, (C, G, K) doxorubicin, and (D, H, L) Au-doxorubicin conjugates. The pink areas indicate necrosis, with individual apoptotic cells appearing black (arrow).
of apoptotic bodies and inflammatory cells was observed (Figure 5F), often occupying the bulk of the tumor volume.
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TUNEL staining of histology sections showed positive “hot spots” in Dox and Au-Dox treated B16 (Figure 6, A-D) and SK-MEL-28 (Figure 6, E-H) tumors. The spots were more intense in the Au-Dox case and represented a greater fraction of the cells than with Dox alone in both types of tumors. Nonetheless, most of the cell death observed with Au-Dox was necrotic.
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It was important to establish that Au-Dox injected into tumors was able to enter the tumor cells themselves, rather than being restricted to cells of the reticuloendothelial system. Because the particles used in this study were so small, they could not be identified by light microscopy without silver enhancement. With silver enhancement, they could be unambiguously distinguished from melanin granules only when present in cell nuclei. Spherical areas 1-2 mm in diameter were found only in Au and Au-Dox treated sections and not in controls or Dox treated sections (Figure 7A). Their presence in B16 nuclei confirmed that Au-Dox could be taken up by tumor cells in vivo, and STEM images were taken to confirm this. Figure 7, B-D show high-resolution STEM images of tumor sections.
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Figure 5. H.E. staining of SK-MEL-28 tumors treated with Dox (A, C, E) or Au-Dox (B, D, F) at low (A, B), medium (C, D) and high (E, F) magnification.
Discussion
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This study examined the in vivo efficacy of Au-Dox conjugates against two types of melanoma models: murine B16 (Dox-resistant) and human SK-MEL-28 (sensitive). We had previously 17 found that the in vitro efficacy of Au-Dox is greatly improved over Dox alone in Dox-resistant cell lines, but less so in Dox-sensitive cells. This result was supported here, where the B16 cells showed a 20-fold reduction in IC50 with Au-Dox vs. Dox alone. SK-MEL-28 cells only showed a 2-fold reduction, but this represented a significant improvement with Au-Dox over Dox alone. One explanation for the improved performance of Au-Dox lies in the uptake mechanisms, studied here by flow cytometry. The Au-Dox conjugates are taken up by at least two kinds of endocytosis (clathrin-mediated and caveolae), while the Dox alone penetrate the cells through simple diffusion — a well-recognized uptake mechanism for Dox. 24,25
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It is less efficient than energy-dependent uptake, and this explains why uptake of Au-Dox is more rapid and more thorough than uptake of Dox alone. In mice, Au-Dox was substantially superior to Dox alone in both types of tumor tested. Because of their large, aggressive nature, B16 tumors are rarely completely cured; however, Au-Dox led to a significant reduction in tumor size vs. control. Histological analysis demonstrated that most of the residual tumor in the Au-Dox treated animals was necrotic, and STEM showed that the tumor cells were able to take up the particles. Dox alone was significantly less effective than Au-Dox at promoting and maintaining tumor regression. Although effective at first, Dox treated tumors eventually began growing rapidly again, whereas those treated with Au-Dox showed long term growth inhibition. The best results were obtained with intratumoral injection. Intratumoral therapy is used in melanoma that presents as a large
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primary tumor. Delivery of various therapies has been tested in mouse models via this route: immunotherapy, gene therapy, and micro RNAs. 26–28 Intratumoral delivery of adenoviral gene therapy has shown to be effective in spontaneous canine melanoma. 29 In our study, we found that SK-MEL-28 tumors could be almost completely controlled for N 100 days using weekly injections of Au-Dox. By comparison, Dox alone led to accelerated tumor growth after 40–50 days. Because previous reports have indicated minimal systemic biodistribution of nanoparticles post intratumoral injection, 30–32 we did not measure biodistribution from intratumoral injection. Instead we chose to study any possible adverse effects of Au-Dox on nontarget organs by delivering particles intravenously, which leads to substantial accumulation in liver and kidney. Because we did not see significant pathology in these organs even after IV delivery, we are confident that intratumoral injection poses very little risk of liver and kidney damage.
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Figure 6. TUNEL staining of histology sections. The left panels show fluorescence signal only; the right panels show overlay of fluorescence and DIC. (A) Low magnification image of hot spot in Dox-only treated B16 tumor. (B) High magnification image of hot spot in Dox treated B16 tumor. (C) Low magnification image of hot spot in Au-Dox treated B16 tumor. (D) High magnification image of hot spot in Au-Dox treated B16 tumor. (E) Low magnification image of hot spot in Dox-only treated SK-MEL-28 tumor. (F) High magnification image of hot spot in Dox-treated SK-MEL-28 tumor. (G) Low magnification image of hot spot in Au-Dox treated SK-MEL-28 tumor. (H) High magnification image of hot spot in Au-Dox treated SK-MEL-28 tumor.
Although some hot spots of apoptosis were seen with both Dox and Au-Dox, the primary mechanism of cell death in Au-Dox treated tumors was necrosis. Necrotic cell death is associated with physical insults, failure of osmotic regulation, and ATP depletion, 33,34 all of which are likely to be caused by massive uptake of Au-Dox through energy-dependent mechanisms. Necrosis also leads to inflammatory responses. Consistent with such effects, we observed extensive infiltration of immune cells throughout the SK-MEL-28 xenografts. The role of autophagy was not examined here, although it is a common mechanism of cell death associated with nanoparticle overload. 35–38 Future work in our laboratory will examine the mechanisms of cell death more closely in vitro and in vivo. Two factors limited the use of intravenous delivery in this study. The first was the toxicity of Dox alone, which led to death in 2/3 mice in a pilot study, making it difficult to perform appropriate control experiments. The second factor was the
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limited EPR effect seen after repeated Au-Dox and AuNP injections. After a single injection, a significant fraction of the nanoparticles were found in the tumor (close to 40%); however, with multiple injections over two weeks, microgram amounts of gold were seen in liver and kidney, with sub-therapeutic levels in tumor. Improving the targeting and excretion of particles will be necessary to make Au-Dox a useful agent for IV injection. Improved targeting may be possible by conjugating tumorpenetrating or receptor-binding peptides to the surface of the nanoparticles along with the Dox. One option for receptor targeting in melanoma is the melanocortin receptor, targeted by the tridecapeptide α-MSH (melanocyte stimulating hormone). MSH is produced by the brain and pituitary gland and is the most potent melanotropic peptide in the regulation of skin pigmentation via the MC1R. Radiolabeled α-MSH and its analogs have been used to image melanoma with essentially no uptake in non-target tissues except kidney. 39 Future studies will determine whether specific targeting improves uptake after IV
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Figure 7. Light and EM microscopy of histology sections from B16 tumors after IV injection of Au-Dox. (A) Silver staining. The black arrows indicate the Au. The white arrowhead shows nonspecific dark areas that are found in controls. The inset shows a high-magnification image of a single cell. (B) Low-magnification of STEM section, showing tumor cells, adipocytes (arrowhead), and deposits of Au (arrow). (C) Higher magnification image of B16 cell, showing intracellular and intranuclear Au. (D) STEM image showing Au within a capillary (arrows). (E,F) Cell nuclei in tumors with (E) and without (F) Au-Dox to illustrate the difference in contrast.
injection and whether it leads to greater efficacy after either IV or intratumoral injection. This study represents a promising step towards the development of clinical agents for tumors with native or acquired doxorubicin resistance. The synthesis procedure is simple, easily scaled to large volumes, and does not involve toxic precursors such as cetyltrimethylammonium bromide (CTAB). Tiopronin is FDA-approved for the prevention of kidney stones (trade name Thiola). The biggest hurdle will be producing the gold nanoparticles through a reproducible method and obtaining approval for their use as a drug, though there is significant precedent in this area. Several gold-chemotherapy agents are in clinical trials or seeking FDA approval. CytImmune’s goldtumor necrosis factor (TNF) conjugate, Aurimune, entered Phase I trials in 2006. 40 In the Phase I trial, no toxicity due to the gold was observed in the 16 human subjects. In fact, a reduction in toxicity was seen relative to free TNF, with the typical dose-limiting hypotension not seen with the conjugates. The
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drug is currently in Phase II trials for pancreatic cancer, melanoma, soft tissue sarcoma, ovarian, and breast cancer. Another gold preparation is Nanospectra’s “AuroLase” particles, used for photothermal therapy. 41–43 These particles have been approved for investigational use in refractory head and neck cancer and lung cancer. The greatest barrier to FDA approval of gold nanoparticle preparations for imaging is the slow rate of clearance of larger (10-15 nm) particles. 44 However, smaller particles clear more quickly, and the ultrasmall gold particles we report here were cleared by the kidneys in mice within several hours. The design principles presented here are also generalizable to other chemotherapeutic agents besides doxorubicin. Many traditional chemotherapeutic agents may be more effective when conjugated to nanoparticles, especially in synergy with receptor blockade and/or radiotherapy. 45,46 Progress in this area would open the door to a variety of potential treatment strategies for drug-resistant tumors.
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42. Morton JG, Day ES, Halas NJ, West JL. Nanoshells for photothermal cancer therapy. Methods Mol Biol 2010;624:101-17. 43. Day ES, et al. Vascular-targeted photothermal therapy of an orthotopic murine glioma model. Nanomedicine (Lond) 2012;7:1133-48. 44. Hall JB, Dobrovolskaia MA, Patri AK, McNeil SE. Characterization of nanoparticles for therapeutics. Nanomedicine (Lond) 2007;2:789-803. 45. Starkey JR, et al. New two-photon activated photodynamic therapy sensitizers induce xenograft tumor regressions after near-IR laser treatment through the body of the host mouse. Clin Cancer Res 2008;14:6564-73. 46. Atkinson RL, et al. Thermal enhancement with optically activated gold nanoshells sensitizes breast cancer stem cells to radiation therapy. Sci Transl Med 2010;2:55ra79.
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Graphical Abstract
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Intratumoral gold-doxorubicin is effective in treating melanoma in mice
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Department of Biomedical Engineering, McGill University, Montreal QC Canada Department of Biochemistry, McGill University, Montreal QC Canada Goodman Cancer Research Centre, McGill University, Montreal QC Canada
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Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice.
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