We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.
The overall goal of this method is to prepare nanostructured hybrid particles using carbon nanotubes, or CNTs, and lipid self-assemblies. This method presents one of the smart applications of carbon nanotubes, where the stabilized nanostructure emulsion of internally self-assembled lipid particles. The main advantage of this technique is that the protocol is fairly simple, and it combines element properties of both CNTs and lipid nanostructures.
The CNT stabilization produces an oil and water emulsion where the particles have a size of about one micron or below, and the core contains a bi-continuous cubic lipid nanostructure. Due to coating of CNTs with bio-compatible lipid molecules, their toxicity is anticipated to be low. Recent reports have so far shown that functionalization remarkably reduces toxic, cytotoxic, effects of CNTs while increasing bio-compatibility.
Demonstrating the procedure will be Yogita Patil-Sen a post-doc from my laboratory, and Amin Sadgehpour a post-doc from Professor Rappolt's laboratory. Prepare a 0.2%surfactant solution in water by dissolving 200 milligrams of the surfactant in 100 milliliters of ultra-pure water by stirring it for 20 to 30 minutes. Add 500 milligrams of molten Dimodan U, DU, or Phytantriol, or PT, to a glass vial.
Then add 9.5 grams of the 0.2%F127 solution. On the probe ultrasonication machine, tightly clamp the vial to the retort stand jaw, so that it can withstand the vibrations generated by sonication. Insert the solid titanium alloy probe attached to the cell sonicator.
Adjust the height and position of the vial to ensure that its sides and bottom are not touching to the probe. Sonicate the mixture for ten minutes in a pulsed mode, with a one-second pulse mediated by a one-second delay time at 35%power. The vial gets very hot due to the heat generated during sonication.
Therefore, allow it to cool down to room temperature before taking it off the clamp. Store the milky formed dispersion at room temperature for at least 24 hours prior to further use. This is to ensure its stability against phased separation.
Before and after using the probe clean it with acetone and dry with a paper towel. Then rinse it with ultra-pure water and dry it once more. In two separate beakers, weigh in four milligrams of powdered hydroxy-functionalized CNTs, and carboxyl-functionalized CNTs, both of which are black in color.
Add 500 milliliters of ultra-pure water to each beaker. Using a probe ultrasonicator, sonicate the mixtures for two minutes in a continuous pulse mode at 40%power. The resulting concentration of the multi-walled CNT dispersion is eight micrograms per milliliter.
Dilute the multi-walled CNT stock solution with appropriate amounts of ultra-pure water to achieve multi-walled CNT dispersions at several different concentrations. Sonicate these dispersions as before. Similarly, disperse three milligrams of powdered, single-walled CNTs in 500 milliliters of ultra-pure water to make a six microgram per milliliter single-walled CNT dispersion stock solution.
Dilute the single-walled CNT stock solution and sonicate as before to obtain single-walled CNT dispersions of various concentrations. Weigh 500 milligrams of the molten DU into a glass vial. Then, add 9.5 milliliters of the six microgram per milliliter single-walled CNT dispersion to the vial.
Sonicate the CNT DU mixture using the same parameters as used for making pure CNT dispersions. Upon cooling to room temperature, the CNT-stabilized lipid particles with conserved internally self-assembled nanostructure will be ready. In a similar way, prepare the lipid particles using the 0.4 micrograms per milliliter and 0.2 micrograms per milliliter single-walled CNT dispersions.
Sonicate the CNT PT dispersions at 35%power for a longer time of fifteen minutes in a continuous pulse mode. Cool the dispersions to room temperature, and leave them for 24 hours before characterizing them. Monitor the stability of the dispersions by visual observation.
Check if the dispersions are destabilized, or if lumps have formed in the dispersions. Take photos at regular intervals with a digital camera. For instance, take pictures of dispersions everyday in the first week.
Then every other day for a week. Followed by once a week for the next two weeks, and finally once a month as per requirement. The small-angle Xray scattering technique is used to determine the lattice type of the inter-nanostructure of the stabilized lipid particles.
Fill the sample in a one millimeter quartz capillary, and seal it properly. Then place the capillary in a temperature controlled sample stage. Evacuate the sample chamber using a vacuum pump.
Record the 1D scattering patterns with a micro-strip x-ray detector controlled by software. After obtaining three profiles with an exposure time of 300 seconds, integrate these profiles and proceed with analysis as described in the text protocol. Index all recorded diffraction patterns with a space group PN3M in which the one-one-zero, one-one-one, two-zero-zero, two-one-one, two-two-zero, and two-two-one reflections can be clearly noticed.
Highly viscous lipid cubic phase is defragmented into particles using ultrasonication. Pristine singe-walled, or functionalized multi-walled CNTs are used as stabilizers to produce oil and water emulsion exhibiting fluid consistency. Optimal CNT to lipid ratio is required to obtain homogeneous and stable emulsions.
Emulsions with too little CNTs contain lipid lumps. Whereas emulsions with excess CNTs contain CNT lipid lumps. Small-angle x-ray scattering patterns confirm that the PN3M cubic phase, displayed also by bulk lipid Phytantriol in excess water, is retained in F127 stabilized emulsions, as well as in all CNT-stabilized emulsions.
Static light-scattering studies demonstrate that the size distribution of cubosomes stabilized by F127 is comparable with the size distribution of CNT stabilized cubosomes, and the average particle size lies in the sub-micron range. Characteristic G, and G prime bands, or CNTs observed in Raman spectra, show blue shift for CNT-stabilized lipid particles. This demonstrates possible coating of CNTs by lipid molecules, as represented by this schematic diagram.
After watching this review you should have a good understanding of how to prepare CNT lipid hybrid particles as has been demonstrated in few, easy steps. These nano-structured lipid particles and about learning of function molecules within the lipid self-assembly, as well as on the CNT surface, which paves the way for exploring hybrid nano-molecules in combination therapies against major diseases.
