Development of medical-grade, discrete, multi-walled carbon nanotubes as drug delivery molecules to enhance the treatment of hematological malignancies

Abstract

Carbon nanotubes (CNTs) hold great potential as drug delivery transporters given their large drug-binding surface area. Herein, we designed novel, multi-walled, discrete CNTs (dMWCNTs), PEGylated dMWCNTs (PEG-dMWCNTs), and bone-targeting (BT), alendronate-conjugated PEG-dMWCNTs (BT-PEG-dMWCNTs). Using zeta potential, thermogravimetric analysis, TEM, SEM, and FTIR, dMWCNTs were characterized as individual, uniform, and stable. Drug binding and release assays validated dMWCNTs as effective doxorubicin (DOX) transporters. The mass ratio of DOX loading onto dMWCNTs was 35% wt/wt with a ~95% wt/wt efficiency. DOX release was ~51% w/w after 48 hours. Neoplastic transformation, chromosomal aberration, and cytotoxicity assays, confirmed biocompatibility for all dMWCNTs. PEG-dMWCNTs were well tolerated and modulated drug pharmacokinetics in mice. In mice with Burkitt's lymphoma, DOX-loaded PEG-dMWCNTs and BT-PEG-dMWCNTs reduced tumor burden and increased survival similarly to free drug. Importantly, DOX toxicity was abrogated when DOX was loaded onto PEG-dMWCNTs or BT-PEG-dMWCNTs. Overall, PEG-dMWCNTs and BT-PEG-dMWCNTs represent a promising new nanocarrier platform.

Keywords: Burkitt's lymphoma; Discrete carbon nanotubes; Drug delivery; Multiwalled carbon nanotubes; Nanomedicine.

Figure 1:. Physical Characterization of dMWCNTs.

A) Representative SEM micrographs of entangled CNTs aggregated tubes and individualized dMWCNTs and PEG-dMWCNTs are discrete and substantially free of contaminants (silicon wafer seen in backdrop). B) TEM analysis proves they have an outer diameter of 10.74 nm +/− 2.46 (SD) nm and an inner diameter of 4.959 nm +/−1.07 (SD). C) Molecular spectral properties using FTIR of dMWCNTs and functionalized PEG-dMWCNTs. D) TGA comparison between CNTs and dMWCNTs show an increase in oxidative functionalization and removal of metal catalyst during the cleaning process to yield dMWCNTs from CNTs E) TGA comparison between dMWCNTs and PEG-dMWCNTs show an increase in functionalization due to the presence of PEG covalently attached to the surface.

Figure 2:. Cytotoxicity Profiles of dMWCNTs vs PEG-dMWCNTs.

Cytotoxicity assay comparing multiple cell types over a 24 and 48-hour treatment period with various doses of dMWCNTs and PEG-dMWCNTs (A) OPM2, (B) hMSCs, (C) 3T3L1 cells. Statistics were done using 2-way ANOVA with Sidak’s multiple comparisons test at 24 and 48 hours for each cell line, n=4-6.

Figure 3:. Development of BT-PEG-dMWCNTs.

A) Fluorescent imaging of ex vivo bones samples in which CY5-PEG-dMWCNTs and BT-CY5-PEG-dMWCNTs were incubated with mouse calvarias bone. Scale bar = 100μm. B) Quantification of the ex vivo BT-PEG-dMWCNT binding affinity at 2 hours using RGB (red, green blue) intensity measurements on ImageJ. C) Cy5 fluorescence reading of bone after PEG-dMWCNT and BT-PEG-dMWCNT incubation with bone autofluorescence subtracted out. Data are plotted as mean ± SEM. Statistical significance evaluated with Student’s T-tests where **** represents p<0.0001. D) SEM of BT-PEG-dMWCNTs showed that the tubes maintained their individuality following attachment of alendronate. E) TEM of BT-PEG-dMWCNTs showed no distinguishable difference between PEG-dMWCNTs.

Figure 4.. Characterization of BT-PEG-dMWCNTs.

Surface functionalization of alendronate by FTIR (A), TGA (B), and stability studies (C-D) of bone targeted PEG-dMWCNTs. E) Cell toxicity of BT-PEG-dMWCNTs in 3 cell types after 24 hours (E) and 48 hours (F) showed similar trends with more toxicity after 48 hours. Statistics were done using 2-way ANOVA with Sidak’s multiple comparisons test at 24 and 48 hours for each cell line, n=3.

Figure 5:. Loading and Release Properties of PEG-dMWCNTs and BT-PEG-dMWCNTs.

A) DOX loading efficacy and potential curves indicate PEG-dMWCNT saturation is 35% wt/wt with higher ratios leaving some DOX remaining unbound. B) Percent of Maximum Adjusted DOX Fluorescence showing off-rate analysis as an initial fast release through ~4 hours and a longer release over days. There is no difference between PEG-dMWCNTs and BT-PEG-dMWCNTs DOX release rate. C-F) In Vivo Pharmacokinetic Comparison of Free DOX vs. PEG-dMWCNT-Loaded DOX. C) Curve fit of blood serum [DOX] data over a 48h period from mice. PEG-dMWCNT-loaded DOX remained higher in the crucial first hours of circulation and both curves eventually plateau similarly. D) Area Under the Curve (AUC) calculation shows a doubling of the mouse’s exposure to DOX when PEG-dMWCNT-loaded. E) Calculated half-lives do not show a difference between free DOX and PEG-dMWCNT-DOX and indicates that Free DOX or DOX released from PEG-dMWCNTs are cleared and metabolized similarly. F)Liver DOX concentrations. Data are plotted as mean ± SEM. Statistically significance was evaluated with Student’s T-tests where: **, p=0.0069; ***, p=0.0011.

Figure 6:. In Vitro Efficacy of DOX loaded dMWCNTs in Raji.luc cells.

A) Raji.luc cell survival assay reveals cell death in concentration-dependent manner in response to PEG-dMWCNTs and BT-PEG-dMWCNTs after 24 and 48 hours. 2-way ANOVA and Dunnett’s multiple comparison test was done at each concentration for each time point with significance indicated with * (p>0.05) compared to control. B) Raji.luc cell survival assay reveals cell death in concentration- and time-dependent manner in response to DOX-loaded PEG-dMWCNTs, DOX-loaded BT-PEG-dMWCNTs and free DOX after 24 and 48 hours. Data is represented as mean ± SEM, n=12.

Figure 7:. Treatment of Burkett's Lymphoma with PEG-dMWCNT-Loaded and BT-PEG-dMWCNT-Loaded Doxorubicin.

A) BLI indicates a near complete abrogation of the tumor population in DOX and PEG-dMWCNT-loaded DOX groups and a decrease in tumor burden with PEG-dMWCNTs alone. B) Kaplan-Meier curve analysis showed a near total loss of control animals over the 45-d study but an increase in survival with free DOX, PEG-dMWCNT, PEG-dMWCNT-DOX, and BT-PEG-dMWCNT-DOX groups. Two mice were lost from the free DOX group due to treatment side effects; no animals were lost from either PEG-dMWCNT-loaded DOX group. C) DOX elicits a near 15% decrease in animal weight over 15 d, which was eliminated when DOX was dMWCNT loaded (see Table 2 for statistics). Data are plotted as mean ± SEM.