We have fabricated surface-modified paclitaxel nanowires (SM-PNs) with a precise diameter and an average length of 50
Many promising anticancer drugs have very low solubility in aqueous and biological media [
A general way of preparing drug filled nanoparticles is to inject an ethanol or chloroform solution of the drug and emulsifier (usually a nonionic polymer surfactant such as polyethylene glycol PEG, or Chremophor EL) in an aqueous saline or buffered media [
(a) Drug-loaded nanoparticle with cluster of drug molecules randomly distributed though out the vehicle matrix. Most of the drug is distributed on the surface of the matrix, which leads to the burst effect. (b) SM-PN with an exact diameter and length. The entire drug is located inside the vehicle (ODS shell). The alkyl group R is anchored inside the drug nanowire with the Si–OH sticking out. ODS stands for octadecylsiloxane.
In a previous paper we fabricated surface-modified paclitaxel nanowires (SM-PNs) using anodic aluminum oxide (AAO) templates coated with a monolayer of n-octadecylsilane [
AAO templates were purchased from two separate sources. Templates with a nominal pore diameter of 200 nm (Anodisc-13) were purchased from Whatman Inc., while precision AAO templates with a narrow pore size distribution (80, 35 and 18 nm) were acquired from Synkera Technologies. All the templates had the same diameter of 1.3 cm and approximate template thickness of 50 Preparation of SM-PNs: these nanowires were prepared as described previously [ Preparation of microcrystalline paclitaxel or free paclitaxel (FT): a solution of paclitaxel in THF (1.8 mg in 100 Scanning Electron Microscopy (SEM): a drop of the paclitaxel microcrystalline suspension was filtered through a 20 nm pore diameter AAO template then washed with 0.2 mL deionized (D.I.) water before air-drying at room temperature. The AAO template/paclitaxel microcrystal residue was fixed on an SEM stub via conducting carbon tape and sputter coated with Pt (~5 nm thickness). Fabrication of multilayered ODS shells: an AAO template (Synkera, 80, 35, 18 m) or Whatman (200 nm pore diameter) was sonicated with ethanol then hexane prior to drying in an oven set at 120°C for 2 hours. The template was placed over a fritted funnel and suction applied. A 5% solution of n-octadecyltrichlorosilane (OTS) in hexane was filtered through 0.1 mL at a time followed by 0.1 mL of water-saturated benzene to hydrolyze the Si–Cl bond and form a reactive Si–OH handle. The process was repeated several times till the AAO pores become clogged and the hexane solution wont filter through. The template was air dried, polished with 1500 grit abrasive paper then dried in an oven at 120°C for 2 hours. The multilayered ODS shells were liberated by dissolving the template in 20% H3PO4/0.4% SDS. In Vitro assay: human monocytic U937 cancer cells ( UV-Vis measurement of paclitaxel concentration in PBS solution: A templated sample of paclitaxel in AAO was submerged in a vial containing 25 mL of PBS. The vial was placed in a water bath set at 37°C and slowly agitated for two days. A sample of the buffer solution was measured using UV-Vis every 12 hour. To determine the paclitaxel concentrations from the absorption spectrum, absorption coefficient at the peak of the absorption was taken to be (
There are several benefits from using nanowires or 1D nanostructures as drug carriers over nanoparticles or 0-dimensional (0D) nanostructures. Nanowires tend to incorporate inside cells more efficiently than nanoparticles [
As mentioned earlier, the high solubility of paclitaxel in organic solvents makes it amenable to nanowire formation via solvent annealing inside AAO templates with any pore sizes or template thickness. In a previous paper, we employed a stepwise bottom-up approach to fabricate surface-modified paclitaxel nanowires (SM-PNs) using AAO templates with precise pore diameter and template thickness [
(a) SEM image of SM-PNs with 200 nm diameters. (b) SEM image of 80 nm SM-PNs bundles. (c) SEM image of 35 nm SM-PNs over AAO template. (d) Close-up image of a single 35 nm SM-PN over an AAO template. The AAO pores in the background have an average diameter of 40 nm.
Vials containing paclitaxel in 20% H3PO4/0.4% SDS. (a) 580
The tethered surface coating of ODS in SM-PNs presents a unique vehicle where the coating does not have to be above a certain critical micelle concentration (CMC) to surround the drug and thus can be diluted without the risk of dissociating from the surface. The ratio of ODS to drug was determined gravimetrically for each SM-PN diameter. The benefits from the stepwise bottom-up templated approach are a typical paclitaxel loading of 75% for a 35 nm diameter SM-PNs. This is not the case with micelleular-, liposome- or polymer-based drug nanoparticles where encapsulation efficiency and drug loading capacity are usually small and variable [
In order to test whether SM-PNs retain their cytotoxic potential, we studied the effects of these nanowires on U937 cells. To rule out the cytotoxicity of the ODS layer, empty ODS shells of different diameters (200, 80, 35, and 18 nm) were incubated with U937 cells culture for a period of two days. ODS shells had no effect. However, testing empty ODS shells was problematic because these monolayers are too thin and will collapse once they are released from the template because of their hydrophobic core. Collapsed ODS shells might not reflect a clear evaluation of the ODS cytotoxicity in a nanowire form. Therefore the shells were stiffened by casting several layers of OTS (n-Octadecyltrichlorosilane) inside the shell via multiple dip drying cycles [
Number of SM-PNs containing 3
SM-PN diameter (nm) |
*Total number of SM-PNs |
Ratio of SM-PN to U937 cells |
---|---|---|
200 | 2.8 | 2 : 1 |
80 | 19 | 10 : 1 |
35 | 66 | 37 : 1 |
18 | 499 | 277 : 1 |
*The average SM-PN length is ~
The next step was to study the cytotoxicity of different diameter SM-PNs and compare their cytotoxicity to that of free paclitaxel (FT). A bulk suspension of SM-PNs in 20% phosphoric acid and 0.4% SDS was used. On average it requires 5
Scanning electron micrograph of microcrystalline paclitaxel or free paclitaxel (FT) over an AAO template. (a) Microcrystalline paclitaxel aggregate over an AAO template. Scale bare = 10
The SM-PN diameters investigated in this study were 200 nm, 80 nm, 35 nm, and 18 nm. A total of
As shown in Table
In chemotherapy one of the primary goals is to slow down or stop cancer cell proliferation during the early stages of administering the drug. paclitaxel stabilizes tubule polymerization and stops cell division; thus it acts on inhibiting cell proliferation and then inducing apoptosis in cells [
24, 48, and 72 hour incubation periods, respectively.
Time | Parameter | Untreated | FT | SM-PN | SM-PN | SM-PN | SM-PN |
---|---|---|---|---|---|---|---|
200 nm | 80 nm | 35 nm | 18 nm | ||||
24 h | Total cells* (SD) | 3.70 (0.126) | 2.53 (0.12) | 2.28 (0.16) | 2.10 (0.14) | 1.68 (0.15) | 2.81 (0.13) |
Nonviable cells* (SD) | 0.18 (0.006) | 1.12 (0.05) | 1.02 (0.07) | 0.81 (0.05) | 0.71 (0.06) | 1.22 (0.057) | |
% Nonviable (SD) | 4.9 (0.17) | 43.5 (2.07) | 44.0 (3.1) | 38.2 (2.6) | 41.8 (3.6) | 42.8 (2.12) | |
% Proliferation (SD) | 106 (7.03) | 40.7 (6.73) | 26.8 (8.90) | 16.7 (7.85) | −6.5 (8.17) | 56 (7.53) | |
| |||||||
Total cells (SD) | 7.98 (0.16) | 2.02 (0.18) | 2.03 (0.22) | 1.78 (0.19) | 1.48 (0.17) | 1.91 (0.15) | |
| |||||||
48 h | Nonviable cells (SD) | 0.40 (0.008) | 1.69 (0.15) | 1.82 (0.20) | 1.50 (0.13) | 1.47 (0.17) | 1.49 (0.12) |
% Nonviable (SD) | 5.0 (0.10) | 84.9 (7.93) | 89.4 (9.7) | 85.0 (9.8) | 100 (12.8) | 79.0 (6.6) | |
% Proliferation (SD) | 344 (8.90) | 12.0 (10.2) | 13.0 (12.5) | −0.9 (10.8) | −17.6 (9.5) | 6.0 (8.7) | |
| |||||||
Total cells (SD) | 9.48 (0.18) | 1.70 (0.24) | 1.32 (0.19) | 1.70 (0.23) | 1.18 (0.15) | 1.72 (0.18) | |
| |||||||
72 h | Nonviable cells (SD) | 1.30 (0.025) | 1.50 (0.21) | 1.31 (0.19) | 1.69 (0.23) | 1.18 (0.15) | 1.70 (0.18) |
% Nonviable (SD) | 13.7 (0.26) | 89.7 (12.6) | 100 (16.5) | 100 (13.8) | 100 (15.3) | 99.9 (10.4) | |
% Proliferation (SD) | 427 (10.2) | −5.5 (13.1) | −26.8 (10.7) | −5.65 (12.7) | −34.2 (8.2) | −4.6 (10.2) |
SD: standard deviation.
*Total number of nonviable or viable cells
% nonviable cells = (number of nonviable cells)/Total number of cells
% Proliferation = (Total number of cells−initial number of cells)/initial number of cells
Optical microscope images of U937 cells. (a) Untreated U937 cells. The cells tend to be asymmetrical with a dark center. (b) U937 cells treated with FT after 72 hours. Notice the pronounced swelling and appearance of vacuoles (white dots in the middle). (c) U937 cells treated with 200 nm diameter SM-PNs after 72 hours of incubation. Nanowires are visible in the background with the presence of cellular debris. (d) U937 cells treated with 35 nm diameter SM-PNs after 72 hours of incubation. The nanowires are too small to be viewed at this magnification. Cellular debris from burst cells appear as specs in the background. Scale bar = 10
(a, b, c) Total number of U937 cells with different paclitaxel forms after 24, 48, and 72 h, respectively. Dotted line represents the initial number of cells
The solubility experiment has provided some mechanistic evidence that there is no significant dissolution of paclitaxel out of the ODS shell that may inhibit cell proliferation. Therefore, it is possible that SM-PNs should physically contact with the cells in order to affect them. To eliminate the possibility that the tiny amount of dissolved paclitaxel is causing a significant impact on the cell proliferation, we incubated the same number of U937 cells
SM-PNs of different diameters were fabricated and their cytotoxicity on U937 cells was studied in vitro. For the same ratio of paclitaxel/cells, the most efficient SM-PNs had a diameter of 35 nm with a complete inhibition of cell proliferation in the first 24 hours of incubation. Different size nanowires have shown different effects on cell proliferation rate. This difference rules out the possibility that paclitaxel dissolved out of the ODS shell prior to interaction with the cell. It is still an open question concerning the mechanism of action. In this proof-of-concept, we have shown that by utilizing a simple solvent annealing method inside surface-modified AAO templates, we can generate SM-PNs with tailored cytotoxicity using a novel drug delivery vehicle.
R. O. Al-Kaysi acknowledges the support of KSAU-HS/KAIMRC through Grants RC08/093 and RC10/104 and King Abdulaziz City for Science and Technology (KACST) through Grant AT-30-435. The authors greatly acknowledge Professor C. J. Bardeen of the Department of chemistry, University of California Riverside for helpful discussions.