Synthesis of nanodiamond-daunorubicin conjugates to overcome multidrug chemoresistance in leukemia

Abstract

Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains.

From the clinical editor: The authors of this study demonstrate superior treatment properties of resistant leukemia cell lines by utilizing nanodiamond vectors loaded with daunorubicin, paving the way to clinical studies in the hopefully not too distant future.

Keywords: Chemoresistance; Drug delivery; Leukemia; Nanodiamond; Nanomedicine.

Figure 1

(A) An artistic rendering of the response from a resistant cancer cell to treatment from DNR versus ND-R. On the left, ABC transporter proteins are able to detect DNR within the cell and remove it via efflux pumps. On the right, ND-enabled delivery of DNR is able to bypass the efflux pumps. The reversibly-bound DNR is released in the cell and enters the nucleus, causing apoptosis. (B) Cell viability after exposure to varying concentrations of NDs, demonstrating the biocompatibility of NDs, even at higher concentrations.

Figure 2

(A) Images depicting the DNR loading process. The top row shows a sample of NDs processed without DNR and the bottom row shows ND and DNR at a 5:2 mixing ratio. (i) The first panel shows ND and ND:DNR before any treatment. (ii) The second panel shows ND and ND:DNR after the binding environment has been adjusted to 3 mM of NaOH. (iii) The third panel shows the pelleting of ND and ND-R conjugates after centrifugation. (iv) The fourth panel shows the resuspended solution after supernatant from part (iii) was removed and replaced by an equal volume of water. (B) Graph of the DNR loading efficiency for various binding conditions. The red box indicates the optimal binding condition tested and the conditions used for the rest of the study.

Figure 3

Characterization of the ND-R conjugates formed by the process outlined in Figure 2. (A) A graph of the size distribution of ND (solid line) compared with ND-R conjugates (dashed line) obtained using DLS. The increase in size of ND-R indicates the loading of DNR onto the ND surface. The inset images are taken using TEM, to visualize the differences in the surfaces of ND (top) and ND-R conjugates (bottom). The clear boundaries between ND particles are visible in the top image. The bottom image shows the textured surface present on the ND-R conjugates. This organic layer is indicative of the binding of DNR. (B) FTIR spectra of ND (gray line), DNR (red line), and ND-R (purple line). The spectra confirms the loading of DNR onto ND platforms because the ND-R spectra contains peaks that are characteristic of both ND and DNR spectras.

Figure 4

(A) The release profile of DNR from ND-R conjugates under different pH environments. The graph tracks the DNR elution over a period of 72 hours. At pH 4, matching conditions ND-R particles would experience during the endocytic pathway, the elution profile is relatively steady and sustained. (B) A comparison of the gene expression (mRNA) of resistant versus sensitive versions of the K562 cell line for the three major efflux proteins in the ABC transporter family. These efflux proteins have been attributed to causing MDR in many cancers. Consequently, the resistant version of K562 has exhibited an increase in expression. ** p < 0.05

Figure 5

Molecular dynamic simulations of ND and DNR to confirm DNR release from ND carriers. (A) The NDs considered in the model were 4.1 nm in diameter and consisted of 5795 carbon atoms. They were shaped like truncated octahedrons, with 8 hexagonal surfaces and 6 square surfaces with [111] and [100] facet planes respectively. (B) DNR molecules consist of atoms of carbon (aqua), hydrogen (gray), oxygen (red), and nitrogen (blue). (C) The initial structure of a simulated ND-R with 100 DNR molecules bound to the ND surface. (D) DNR release at pH 2. (E) DNR release at pH 4. (F) DNR release at pH 7.

Figure 6

Examining the efficacy of DNR and ND-R by measuring the cellular viability of K562 and K562/DNR after exposure to range of drug concentrations. (A) A summary of the IC-50, or half maximal inhibitory concentrations of DNR and ND-R treatments. ND-R exhibited improved efficacy towards treating resistant K562 as compared with DNR. The resistance of K562/DNR is confirmed by the 4 fold increase over K562 in IC-50 value when exposed to DNR. **p < 0.5 (B) K562 + DNR (avg. r² = 0.97) (C) K562/DNR + DNR (avg. r² = 0.96) (D) K562 + ND-R (avg. r² = 0.94) (E) K562/DNR + ND-R (avg. r² = 0.96) dose response curves fitted to a sigmoid function. Each graph contains three trials with separate sigmoidal curves fitted to each trial. Mean IC-50 values are averaged over these three trials.