Name: facile preparation of internally self-assembled lipid particles stabilized by carbon nanotubes
Uploaded: 2016-02-19T13:00:00
Description: Watch this Scientific Journal Video about Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes at JoVE.com

We would like to thank Dr. Matthew J. Baker, now at the University of Strathclyde, Glasgow for the support with Raman experiments and Mr. Nick Gaunt for his prior work of this project.

Caution: CNTs used in this work are in the nanoparticulate form which may have additional hazards compared to their bulk counterparts. Inhalation of graphite, both natural and synthetic, can cause pneumoconiosis62 similar to coal worker&#39;s pneumoconiosis. Moreover, there have been concerns relating to the toxicity of carbon based nanostructures and some of the previous studies suggest acute and chronic toxicity associated with the inhalation of CNTs63-68. Hence, avoid inhalation of the fine CNT powder and handle it w...

Stabilization of lipid particles 
Three different CNTs are used to stabilize the lipid dispersions; two of which are multi-walled and functionalized using -OH and -COOH groups, and one is single walled and non-functionalized (pristine). The CNTs varied in size as follows (diameter x length): MWCNT-COOH: 9.5 nm x 1.5 &#181;m; MWCNT-OH: 8-15 nm x 50 &#181;m; SWCNT: 1-2 nm x 1-3 &#181;m. The powdered CNTs were dispersed in water by probe ultra-sonication. Aforementioned sizes of CNTs are likely to decr...

Over the last few decades, nanotechnology has emerged as a powerful tool especially in the field of preclinical development of medicine to combat notorious diseases such as cancer1. In this context, nanoscale structures with size &#60;1,000 nm are extensively explored as delivery vehicle of various active biomolecules such as drugs, proteins, nucleic acids, genes and diagnostic imaging agents1-4. These biomolecules are either encapsulated within the nanoparticles or conjugated onto the surface of nanoparticles and are released at the site of action by triggers such as pH or temperature5,6. Although extremely small in size, the large surface area of these nanoparticles proves to be greatly advantageous for targeted delivery of active biomolecules. The control over the particle size and biocompatibility is of utmost importance in order to optimize the therapeutic efficacy and hence the applicability of nanoparticles7,8. Lipids9-13, polymers14,15, metals16,17 and carbon nanotubes18,19 have been commonly employed as nanocarriers for various biomedical and pharmaceutical applications.
Moreover, nanocarrier applications based on lipid self-assembled nanostructures have a wide significance in many other disciplines including food and cosmetic industries20,21. For instance, they are used in protein crystallization22, separation of biomolecules23, as food stabilizers e.g., in desserts24, and in the delivery of active molecules such as nutrients, flavors and perfumes25-31. Self-assembled lipid nanostructures not only have the ability to release bioactive molecules in a controlled and targeted fashion32-38 but they are also able to protect the functional molecules from chemical and enzymatic degradation39,40. Although planar fluid bilayer is the most common nanostructure formed by amphiphilic lipid molecules in presence of water, other structures such as hexagonal and cubic are also commonly observed20,41,42. The type of nanostructure formed depend upon the lipids&#39; molecular shape structure, the lipid composition in water as well as on the physico-chemical conditions employed such as temperature and pressure43. The applicability of non-planar lipid nanostructures especially that of cubic phases, is restricted because of their high viscosity and non-homogeneous domain consistency. These problems are overcome by dispersing the lipid nanostructures in large amount of water to form oil-in-water (O/W) emulsions containing micron or submicron sized lipid particles. In this manner, a suitable product of low viscosity can be prepared while retaining the original lipid self-assembled structure inside the dispersed particles. The formation of these internally self-assembled particles (abbreviated as ISAsomes44 e.g., cubosomes from cubic phases and hexosomes from hexagonal phases) commonly requires a combination of an high energy input step and the addition of stabilizers such as surfactants or polymers. Recent research in this direction demonstrates the application of various solid particles45 including silica nanoparticles46, clay47-49 and carbon nanotubes50 for the stabilization of aforementioned emulsions, suitably termed as Pickering51 or Ramsden-Pickering emulsions52.
In recent years, carbon based nanostructures such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs) and fullerenes have received a great deal of attention as novel biomaterials53,54. The main concerns are their toxicity55-58, water insolubility59 and hence their biocompatibility56. An efficient way to tackle these issues is the surface functionalization using non-toxic and biocompatible molecules such as lipids. In presence of water, lipids interact with CNTs in a manner that hydrophobic surface of CNTs is shielded from polar aqueous medium whereas the lipid hydrophilic head groups aid their solubility or dispersion in water60,61. Lipids are integral constituents of cellular organelles as well as some food materials, therefore their decoration should ideally decrease the in vivo toxicity of CNTs. Biomedical applications based independently on CNTs18,19 and lipid nanostructures9-13 are under extensive development but the applications that combine properties of the two are not yet well-explored.
In this work, we employ two different types of lipids and three types of CNTs of which SWCNTs are in the pristine form whereas MWCNTs are functionalized with hydroxyl and carboxylic groups. We have used very low concentrations of CNTs to prepare the dispersions whose stability depends upon several factors e.g., the type of lipid, type of CNT, ratio of lipid to CNT used, as well as on the sonication parameters employed such as power and duration. This video protocol provides technical details of a method of kinetically stabilizing lipid nanoparticles using various CNT-stabilizers.

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			<td height="21" style="height:21px;width:281px;">Dimodan U</td>
			<td style="width:312px;">Danisco</td>
			<td style="width:119px;">15312</td>
			<td style="width:756px;">Store at 4&#176;C, Non-hazardous. Irritant to eyes and skin</td>
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			<td>Store at 4&#176;C, Non-hazardous. Irritant to eyes and skin</td>
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			<td height="21" style="height:21px;">Single walled Carbon Nanotubes (90%)</td>
			<td>Nanostructured &#38; Amorphous Materials, Inc.&#160;</td>
			<td>1246YJS</td>
			<td>Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system</td>
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			<td height="84" style="height:84px;width:281px;">Multi-walled carboxylic acid functionalised Carbon Nanotubes (&#62; 80% Caron basis, &#62; 8% carboxylic acid functionalized)</td>
			<td style="width:312px;">Sigma-Aldrich Co. LLC&#160;</td>
			<td style="width:119px;">755125</td>
			<td>Store at room temperature. Away from direct light. Causes serious eye irritation. May cause respiratory irritation</td>
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			<td height="63" style="height:63px;width:281px;">Graphitized Multi-walled hydroxy functionalised Carbon Nanotubes (99.9%)</td>
			<td style="width:312px;">Nanostructured &#38; Amorphous Materials, Inc. (NanoAmor)&#160;</td>
			<td style="width:119px;">1224YJF</td>
			<td>Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system</td>
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			<td height="21" style="height:21px;width:281px;">Pluronic F127</td>
			<td style="width:312px;">Sigma-Aldrich Co. LLC&#160;</td>
			<td style="width:119px;">P2443</td>
			<td>BioReagent, suitable for cell culture. Not a hazardous substance or mixture. Store at room temperature.</td>
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			<td height="21" style="height:21px;width:281px;">Acetone (99.5%)</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
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			<td>Highly flammable liquid. Causes serious eye irritation. May cause drowsiness or dizziness</td>
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			<td style="width:312px;">VWR International Ltd</td>
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			<td>Soda&#8208;lime glass vial with low background count Fitted with foil lined urea cap, 20 ml</td>
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			<td style="width:312px;">VWR International Ltd</td>
			<td>216-3308</td>
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			<td height="21" style="height:21px;width:281px;">Beaker , 1000mL</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">12942161</td>
			<td>heavy duty, low form, with spout and graduations</td>
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			<td height="42" style="height:42px;width:281px;">Pasteur glass pipette (150 mm length) with latex bulb</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">10006021</td>
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			<td height="42" style="height:42px;width:281px;">Microcentrifuge tube conical snap cap 1.5mL</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">11558232</td>
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			<td height="21" style="height:21px;width:281px;">Spatula</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">11352204</td>
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			<td height="21" style="height:21px;width:281px;">Heating magnetic stirrer</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">11715704</td>
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			<td height="42" style="height:42px;width:281px;">Magnetic stirrer bars (cylindrical, opaque PTFE, 30mm x 7mm (l x diameter))</td>
			<td style="width:312px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">10011792</td>
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			<td height="21" style="height:21px;width:281px;">Needle (0.9 mm x 40 mm cannula length)</td>
			<td>Terumo UK Ltd</td>
			<td>MN-2038MQ</td>
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			<td height="42" style="height:42px;width:281px;">Retort Stand Set - With stand, clamp, base, rod, rubber 3 jaw and bosshead</td>
			<td style="width:312px;">Camlab Ltd, UK</td>
			<td>1177157</td>
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			<td style="width:312px;">Barnstead Nanopure, Thermoscientific, USA</td>
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			<td height="43" style="height:43px;width:281px;">Progen Genfuge 24D Digital Microcentrifuge</td>
			<td style="width:312px;">Progen Scientific</td>
			<td>C-2400</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Probe ultra-sonicator, with 13 mm&#160;</td>
			<td>SONICS, Vibracell,&#160; USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5MP camera with auto-focus and LED flash</td>
			<td>Samsung Galaxy Fame Mobile camera</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Raman Spectrometer</td>
			<td style="width:312px;">Horiba Jobin-Yvon LabRAM HR800 spectrometer</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:281px;">Mastersizer 3000</td>
			<td colspan="2">&#160;Malvern Instruments Ltd, Malvern, United Kingdom</td>
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			<td height="21" style="height:21px;">Small angle X-ray scattering (SAXS)</td>
			<td colspan="3">SAXSpace camera (Anton Paar, Graz, Austria), X-ray generating equipment (ISO-DEBYEFLEX3003, GE Inspection Technologies GmbH), closed water circuit (Chilly 35, HYFRA, Germany).&#160;</td>
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facile preparation of internally self-assembled lipid particles stabilized by carbon nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

The following results represent a) the stability of dispersions, b) the size distribution of lipid particles, c) the type of self-assembly and d) the evidence for lipid coating of the CNTs. The stability of dispersions (Figure 2) was monitored using a 5 MP camera with auto-focus and LED flash.
<img alt="Figure 2" src="/files/ftp_upload/53489/53489fig2.jpg" /> 
Figure 2. Schematics of CNT types (A) M...

Watch this Scientific Journal Video about Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes at JoVE.com

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

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.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

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