As-grown Hipco SWNTs functionalized by PEGylated phospholipid(14
) were used, made by sonication of SWNTs in a water solution of phospholipid-PEG and centrifugation to remove large bundles and impurities. The length distribution of the SWNTs was 20-300nm with a mean of ~100 nm (Supplementary Fig. S1
) The PEG functionalized SWNTs exhibited excellent stability without agglomeration in various biological media including serum.(14
) We used branched PEG chains for functionalization of SWNTs (see Method) to afford more functional amine groups at the PEG termini for efficient drug conjugation.(22
) Paclitaxel was conjugated at the 2′-OH position (23
) to the terminal amine group of the branched PEG on SWNTs via a cleavable ester bond (see Method), forming a SWNT-PTX conjugate highly soluble and stable in aqueous solutions (). The un-conjugated paclitaxel was removed thoroughly from the SWNT-PTX solution by filtration. The loading of paclitaxel on SWNTs was characterized to be ~150 per SWNT with ~100nm length by radiolabeling method using tritium (3
H) labeled paclitaxel and a UV-VIS-NIR optical absorbance (, see Method). Dynamic light scattering showed hydrodynamic size of SWNTs before and after PTX conjugation of 120.6 nm and 132.2 nm, respectively, suggesting no significant aggregation of nanotubes after conjugation of hydrophobic drug molecules. The SWNT-PTX conjugate was found stable in physiological buffers with little drug release within 48 hours (Supplementary information, Fig. S2
). In mouse serum, the release of PTX is faster but SWNT-PTX is still stable for hours (Fig. S2
), which is much longer than the blood circulation time of SWNT-PTX as described later. In vitro cell toxicity tests performed with a 4T1 murine breast cancer cell line found that SWNT-PTX exhibited similar toxicity as Taxol® and PEGylated PTX () without any loss of cancer cell destruction ability. Confocal fluorescence images indicated the endocytosis mechanism of the SWNT-PTX uptake by cells (Supplementary information, Fig. S3
). Consistent to the previous studies, (7
) no noticeable toxic effect to cells was observed for plain nanotube carriers without drug even at high SWNT concentrations (Supplementary information, Fig. S4
We next moved to the in vivo
cancer treatment on the paclitaxel resistant 4T1 murine breast cancer mice model.(24
) Female Balb/c mice bearing subcutaneously inoculated 4T1 tumors were treated with different forms of paclitaxel over several weeks including the clinical Taxol® formulation, PEGylated PTX (PEG-PTX, see Method), DSEP-PEG conjugated PTX (DSPE-PEG-PTX) and SWNT-PTX (14 mice in this group). The treatments were done by injecting Taxol®, PEG-PTX, DSEP-PEG-PTX and SWNT-PTX (at the same PTX dose of 5mg/kg for all three formulations, once every 6 days) intravenously into tumor-bearing mice. The mice were observed daily for clinical symptoms and the tumor volume was measured by a digital caliper every other day. As shown in , a time-related increase in tumor volume was observed in the control untreated group and SWNT vehicle only group in which the tumors showed average fractional tumor volumes (V/V0
) of 10.1 ± 1.7 and 9.8 ± 2.0, respectively on day 22. Taxol® treatment, PEG-PTX treatment and DSPE-PEG-PTX treatment resulted in V/V0
of 7.3 ± 1.5 (P = 0.06 vs untreated), 8.0 ± 1.6 (P = 0.18 vs untreated), 8.6 ± 0.9 (P = 0.33 vs untreated) on day 22, which represents tumor growth inhibition (TGI) of 27.7%, 20.8% and 14.9% respectively. In contrast, SWNT-PTX treatment resulted in a V/V0
of 4.1 ± 1.1 on day 22 (P = 2.4×10-6
vs untreated, P = 0.00063 vs Taxol®, P = 0.00026 vs PEG-PTX, 2.7×10-5
vs DSEP-PEG-PTX), representing a TGI of 59.4 %, which is significantly more effective than Taxol®, PEG-PTX and DSPE-PEG-PTX.
To investigate the tumor suppression mechanism, we performed terminal transferase dUTP nick end labeling (TUNEL) assay to examine the apoptosis level in the tumors(26
) from mice received different treatments. Similar to untreated tumor, Taxol® treated tumor showed only 2-3% of apoptotic cells ( 1st
rows, supplementary Fig. S5a
). In contrast, high apoptosis level (~70%, P<0.0001 vs untreated and Taxol® treated tumors) was observed in SWNT-PTX treated tumor (, 4th
row, and see supplementary Fig. S5a
for quantitative comparison), consistent with the improved tumor growth inhibition efficacy (). The Ki-67 antibody staining method has been widely used as a cell proliferation marker to stain proliferation active cells in the G1, G2 and S phases of cell cycle.(27
) We found that cell proliferation in Taxol® treated tumor was as active as in untreated tumor ( 2nd
row, see supplementary Fig. S5b
for quantitative comparison). In the SWNT-PTX treated tumor case however, only ~20% of proliferation active cells were noted compared with the number in the untreated tumor ( 3rd
row, Fig. S5b
, P<0.0001 vs untreated and Taxol® treated tumors). As the control, plain SWNT without PTX showed no effect to the tumors (, 3rd
row), proving the treatment efficacy of SWNT-PTX is due to PTX carried into tumors by nanotubes. Thus, both TUNEL staining and Ki67 staining results clearly confirmed the treatment efficacy of SWNT-PTX by inhibiting proliferation and inducing apoptosis of tumor cells.
Figure 3 Tumor staining for understanding of treatment effects. a, TUNEL (apoptosis assay) and DAPI (nuclear) co-staining images of 4T1 tumor slices from mice after different treatments indicated. While tumors from untreated mice (1st row), Taxol® treated (more ...)
To investigate the pharmacokinetics of various drug complexes, we first measured blood circulation behaviors of PEGylated SWNTs with and without PTX conjugation by Raman spectroscopic detection of SWNTs in blood sample drawn from mice post injection (p.i.) of SWNT and SWNT-PTX (see Method). We observed a significantly shortened circulation half life of our branch-PEGylated SWNT from ~3.3 h to ~1.1 h (circulation half life was obtained by one compartment first order exponential decay fitting, see Method) after PTX conjugation (). This result was important and attributed to the high hydrophobicity of conjugated paclitaxel, reducing the biological inertness of the PEGylated nanotubes in vivo and shortening the blood circulation time. Blood circulation behaviors of the three forms of PTX were measured using 3
H labeled paclitaxel. Liquid scintillation counting of 3
H-PTX radioactivity of blood samples collected from mice post-injection showed circulation half lives of 18.8 ± 1.5 min, 22.8 ± 1.0 min and 81.4 ± 7.4 min for 3
H-PTX injected in Taxol®, PEG-PTX and SWNT-PTX respectively (, see supplementary table S1
for complete pharmacokinetic data). This clearly revealed that conjugation of PTX to PEGylated SWNTs significantly increased the blood circulation time of PTX. Interestingly, simple PEGylation of PTX, though imparted water solubility of PTX, still exhibited much shorted blood circulation than PTX on PEGylated SWNTs. Note that for SWNT-PTX, circulation curves of radiolabeled PTX measured by radioactivity ( green curves) and the drug carrier SWNT measured by Raman have consistent slopes ( red curve), suggesting that PTX and SWNT remained in a conjugated form in the blood circulation stage, which is consistent to the relatively slow PTX releasing behavior of SWNT-PTX in mouse serum (Supplementary information, Fig. S2
). The minor difference in the absolute values could be due to systematic errors between two different methodologies.
Figure 4 Pharmacokinetics and biodistribution. a, blood circulation data of SWNT with and without PTX conjugation (marked as SWNT-PTX (R) and SWNT only (R) respectively) measured by Raman detection of SWNTs in blood samples (see Methods). Blood circulation data (more ...)
To understand the tumor treatment efficacy of various PTX formulations, i.e., SWNT-PTX, Taxol® and PEG-PTX, we investigated biodistribution of 3H-PTX in the tumor and various main organs. We observed significant differences in the biodistribution of PTX administrated in the three formulations of PTX (). Consistent with the blood circulation data (), SWNT-PTX showed noticeable PTX activity in blood at 2h p.i., while PTX levels in the blood were much lower in the Taxol® (P < 0.001) and PEG-PTX (P < 0.01) cases ( insert). Differences in biodistributions of PTX in the three cases were the most obvious at 2h p.i., with much higher PTX signals in the RES organs (liver/spleen) and intestine of mice in the SWNT-PTX case than the two other cases ().
Importantly, SWNT-PTX afforded much higher PTX uptake in the tumor than Taxol® and PEG-PTX. The tumor PTX levels in the SWNT-PTX case was higher than those of Taxol® and PEG-PTX by 10 and 6-fold respectively at 2h p.i. (), and by 6 and 4-fold higher respectively at 24 h p.i. (, P<0.001 in all cases). The ability of higher drug delivery efficiency to tumor by our PEGylated SWNTs was striking and directly responsible for the higher tumor suppression efficacy of SWNT-PTX than the other formulations. This suggests that to reach similar tumor uptake of drug, much lower injected dose can be used by SWNT delivery than Taxol®, which is highly favorable for lowering toxic side effect to normal organs and tissues. An important gauge to drug delivery efficiency is the tumor-to-normal organ/tissue PTX uptake ratios (T/N ratios). We obtained significantly higher T/N PTX uptake ratios (for tumor over liver, spleen, muscle and other organs examined) in the case of SWNT-PTX than Taxol® and PEG-PTX (except at 2 h p.i. for spleen) at the 2 h and 24 h (supplementary table S2
). This again makes SWNT-PTX highly favorable for high tumor suppression efficacy and low side effects.
We investigated the biodistribution of SWNTs injected as SWNT-PTX conjugates into mice by utilizing their intrinsic Raman scattering properties without relying on radio or fluorescent labels.(18
) We observed high uptake of SWNTs in the reticuloendothelial systems (RES)(18
) including liver and spleen (). Tumor uptake of SWNT-PTX increased significantly from ~1%ID/g at 30 min to ~5%ID/g at 2h, indicating accumulation of SWNT-PTX during this period through blood circulation (see for circulation curve). Tumor uptake of SWNTs at 4.7% (std. = 2.1%, n = 3) ID/g was observed at 2h p.i. (), reasonably consistent with the ~6.4% (std. = 1.1%, n = 3) ID/g PTX tumor uptake (), suggesting that SWNT-PTX was taken up by tumor in a conjugated form. The SWNT biodistribution exhibited little change from 2h () to 24h p.i. (), in contrast to the biodistribution of radiolabeled PTX ( green bars). This suggests that the dissociation of PTX from SWNT carriers in vivo resulted from in vivo cleavage of the ester bond between SWNT and PTX is likely by carboxylesterases.(29
Figure 5 SWNT biodistribution measured by Raman spectroscopy. a - c, comparison of 3H-PTX biodistribution and SWNT biodistribution in mice injected with SWNT-PTX(3H) at (a) 30 min, (b) 2 h and (c) 24 h p.i. SWNT biodistribution was measured by a Raman method (see (more ...)
We carried out micro-raman imaging of SWNTs in tumor slices upon sacrificing mice treated by SWNT-PTX at 24 h p.i. The tumor uptake of SWNTs was indeed confirmed by Raman mapping of the SWNT characteristic G-band Raman peak at ~1580 cm-1 in the tumor with a spatial resolution of ~ 1μm (, inset). To investigate the location of nanotubes in the tumor relative to the vasculature, we injected Alexa Fluor 488 (AF488) fluorescently labeled SWNTs into 4T1 tumor bearing mice, sacrificed the mice, collected the tumors for vasculature staining and fluorescence imaging (, right). We observed fluorescently labeled SWNTs both with and without overlaying with tumor vasculatures. This suggested that while most SWNTs appeared to be located in or near the tumor vasculature, a fraction of nanotubes could leak through the tumor vessel into the tumor interstitial space. As the control, no tumor retention of fluorescent dye was observed in mice injected with free AF488 at the same dose (, left).
Toxic side effects to normal organs and overall well being have been the main problems of cancer chemotherapeutics. By themselves, our well PEGylated SWNTs have been found to be non-toxic to mice in vivo monitored over many months.(21
) We carried out a pilot toxicity study by treating healthy, tumor-free Balb/c mice with Taxol® and SWNT-PTX at the same 5mg/kg PTX dose once every six days. We observed neither mortality nor noticeable body weight loss of the mice treated with SWNT-PTX and Taxol® compared to untreated control group at this relatively low PTX dose and injection frequency (). Blood chemistry test was performed 24 days after initiation of the treatment, showing no physiologically significant difference among the 3 groups ( & Supplementary Table S3
). Furthermore, hematoxylin & eosin (H&E) stained sections of the 25 organs and organ systems were examined (), without noticing obvious abnormal damage in the main organs including the liver and spleen that had high SWNT uptake, which was consistent to the normal hepatic enzyme levels measured in the blood chemistry test (). The observed lack of obvious toxic side effect was partly due to the low dose of PTX used as the maximum tolerable dose of PTX in the Taxol® case ~20-50mg/kg.(32
) Achieving tumor treatment efficacy by SWNT-PTX at a PTX dose well below the toxic limit is owed to ability of drug delivery to tumors by SWNTs. However, further careful studies such as the hepatic macrophage function tests are required to examine any potential near-term or long-term side effect our SWNT-PTX.
Figure 6 Pilot toxicity study. a, body weight curves of mice received different treatments in the study (PTX dose~5mg/kg). No obvious loss of body weight was observed in all the groups. 5-14 mice were used in each group (see details in caption). (more ...)