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

Podsumowanie

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.

Streszczenie

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' 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.

Wprowadzenie

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 <1,000 nm are extensively explored as delivery vehicle of various active biomolecules such as drugs, proteins, nucleic acids, genes and diagnostic imaging agents^1-4. 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 temperature^5,6. 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 nanoparticles^7,8. Lipids^9-13, polymers^14,15, metals^16,17 and carbon nanotubes^18,19 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 industries^20,21. 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 perfumes^25-31. Self-assembled lipid nanostructures not only have the ability to release bioactive molecules in a controlled and targeted fashion^32-38 but they are also able to protect the functional molecules from chemical and enzymatic degradation^39,40. 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 observed^20,41,42. The type of nanostructure formed depend upon the lipids' 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, clay^47-49 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 biomaterials^53,54. The main concerns are their toxicity^55-58, 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 water^60,61. 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 CNTs^18,19 and lipid nanostructures^9-13 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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Protokół

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'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 CNTs^63-68. Hence, avoid inhalation of the fine CNT powder and handle it with great care. If inhaled, move to fresh air. If breathing is difficult, use pure oxygen instead and seek medical consultation. Solution/dispersion formulations of CNTs are rather safe to handle.

Caution: Lipids and surfactants used in this study are food-grade materials and thus non-hazardous in general, but they are irritant to eyes and skin, and also highly flammable. Hence, please use all appropriate safety practices such as use of engineering controls (fume hood) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes) when handling or preparing nanoparticle samples. In case of contact with skin or eyes, immediately flush skin or eyes with plenty of water for at least 15 min. Seek medical advice if required.

1. Preparation of Lipid/Water Bulk Phases

Caution: Store the lipids in the refrigerator at 4 °C. Pure grade lipids should be stored in the freezer (-20 °C). Aliquot them into small glass vials to avoid contamination of the whole stock and convenience of handling. Other chemicals including CNTs and surfactants can be stored at RT but keep them away from direct sunlight.

1. Keep lipids, i.e., Dimodan U (DU) and Phytantriol (PT) at RT for 15-20 min prior to opening the bottle/vial lid in order to avoid moisture condensation.
   (Note: DU is a distilled glyceride comprising 96% monoglycerides and the rest are diglycerides and free fatty acids. Two major monoglyceride components in DU are linoleate (62%) and oleate (25%). Hence the hydrophobic part of DU mainly contains C18 chains (91%), the exact composition of which is as follows; C18:2 (61.9%), C18:1 (24.9%), and C18:0 (4.2%), where C18 indicates 18C-chain and the number after colon indicates number of C=C bonds. PT is a mixture of 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol optical isomers. It does not contain an ester functional group but consists of highly branched phytanyl tail with a tri-hydroxy headgroup. Both of DU and PT form cubic phases in presence of excess water which is also the case for the cores of stabilized lipid particles^{13, 45}).
2. Melt the lipids by putting vials in a hot water bath or a beaker containing water maintained above 60 °C (heating magnetic stirrer: 230 V, 50 Hz, 630 W or similar to be used to heat the water in a beaker).
3. Alternatively heat vials using block heaters. Do not heat the lipid containing vials directly on the hot plate in order to avoid temperature gradient and subsequent lipid decomposition.
4. Weigh 500 mg of the molten lipid, in previously weighed microcentrifuge tube (with conical snap cap, 1.5 ml), using a Pasteur glass pipette with a latex bulb.
5. Add 500 ml of ultrapure water (water resistivity=18.2 MΩ·cm) to the above microcentrifuge tube.
6. Mix the components manually for 15 min using tiny (custom-built) spatula. Make such a spatula by flattening the sharp end of a syringe needle (0.9 mm x 40 mm cannula length) using a plier.
7. Centrifuge the lipid/water mixture for 10 min at a speed of 2,000 x g. Again stir the mixture manually for 10 min, then equilibrate it for 24 hr. Before characterizing the samples, stir them for 5 min and then leave them at RT.
8. To ensure the formation of an equilibrium lipid phase throughout the entire tube, perform about 10 freeze-thaw cycles and intermittently carry out a centrifugation step as defined above. Both the DU and PT form highly viscous bulk lipid phases making it difficult to handle them manually (Figure 1).
   Note: The above protocol (section 1) is only necessary, if one would like to compare nanostructural behavior (lattice type and dimensions of self-assembly) of dispersed particles with the bulk lipid phase and/or use it as a control to confirm the retention of original nanostructure.

Figure 1. Preparation of O/W particulate emulsion with fluid consistency from highly viscous lipid phase using high energy input (ultrasonication) and using different CNT-stabilizers, namely SWCNT, MWCNT-OH, MWCNT-COOH (figure reproduced from reference [50] with permission from The Royal Society of Chemistry). Please click here to view a larger version of this figure.

2. Preparation of Surfactant Stabilized Lipid Particles

1. Prepare a 0.2% (w/w) surfactant (pluronic F127) solution in water.
   1. Dissolve 200 mg of the surfactant (white fluffy powder) in 100 ml ultrapure water by stirring it for 20-30 min (on a magnetic plate using a magnetic stirrer bar). Pluronic F127 is a non-ionic surfactant and is commonly used as emulsion stabilizer. It is a triblock copolymer of PEO₉₉ -PPO₆₇-PEO₉₉, and hence takes long time to dissolve in water.
2. Add 500 mg of molten DU or PT (using a Pasteur glass pipette) to a glass vial (scintillation soda-lime fitted with foil lined urea cap, 20 ml).
3. Add 9.5 g of the 0.2% F127 solution.
4. On the probe ultrasonication machine, tightly clamp the vial to the retort stand jaw (Retort Stand Set with stand, clamp, base, rod, rubber 3 jaw and bosshead), so that it can withstand the vibrations generated by sonication.
5. Insert the solid titanium alloy probe (13 mm diameter x 139 mm length) 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. A distance of 0.5 cm between the probe tip and the bottom of the glass vial gives good results.
6. Sonicate the mixture for 10 min in a pulsed mode with 1 sec pulse mediated by 1 sec delay time at 35% (of the maximum) power. The vial becomes very hot due to the heat generated during sonication. Therefore, allow it to cool down to RT before taking it off the clamp.
7. Store the milky-formed dispersion at RT for at least 24 hr, prior to further use. This is to ensure its stability against phase separation.
   Note: Before and after using the probe, clean it with acetone, dry with a paper towel, then rinse it with ultrapure water and dry it once more.

3. Preparation of Dispersions of Pure CNTs in Water

1. In two separate beakers, weigh in 4 mg powdered MWCNT-OH and MWCNT-COOH, both of which are black in color.
2. Add 500 ml ultrapure water to each beaker. Using a probe ultrasonicator sonicate the mixtures for 2 min in a continuous pulse mode at 40% (of the maximum) power. The resulting concentration of the MWCNT dispersion is 8 µg/ml (stock solution).
3. Dilute the MWCNT stock solution with appropriate amounts of ultrapure water to achieve 6.25, 5, 4, 2 µg/ml MWCNT dispersions.
4. Sonicate these dispersions as described before (see 3.2).
5. Similarly, disperse 3 mg of powdered SWCNT (also black in color) in 500 ml ultrapure water to make a 6 µg/ml SWCNT dispersion (stock solution).
6. Dilute the SWCNT stock solution and sonicate them as described above (see 3.2) to obtain 0.5, 0.4, 0.3125, 0.2 µg/ml SWCNT dispersions.
   Note: All dispersions are clear for about 30 min, after which the CNTs start to settle at the bottom.

4. Preparation of CNT-stabilized Nanostructured Lipid Particles (Figure 1)

1. Weigh in 500 mg of the molten DU into a glass vial.
2. Add 9.5 ml of the 6 µg/ml SWCNT dispersion to the vial.
3. Sonicate the CNT-DU mixture using the same parameters as used for making pure CNT dispersions (see 3.2). Upon cooling to RT, the CNT-stabilized lipid particles with conserved internally self-assembled nanostructure will be ready.
4. In a similar way, prepare the lipid particles using the 0.4 µg/ml and 0.2 µg/ml SWCNT dispersions.
5. Follow the protocols 4.1 to 4.4 to make lipid particles using MWCNT-OH and MWCNT-COOH but using different concentrations, namely 8, 4 and 2 µg/ml of CNT.
6. Similarly, prepare three different CNT-PT dispersions using 4 µg/ml MWCNT-OH and MWCNT-COOH as well as 0.4 µg/ml SWCNT. Note that the CNT-PT dispersions require less power (35% of the maximum) but longer time (15 min) in a continuous pulse mode. Cool the dispersions to RT and leave them for 24 hr before characterizing them.
   Note: Sonication parameters may differ for different lipids (as for DU and PT here) and for different compositions; they need to be optimized to achieve well-stabilized dispersions.

5. Monitoring the Stability of the CNT-stabilized Lipid Dispersions

1. Monitor the stability of the dispersions by visual observation: check if the dispersions are destabilized or if lumps have formed in the dispersions.
2. Take photos (with digital camera) at regular intervals. For instance, take pictures of dispersions every day 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.

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Wyniki

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.

Figure 2. Schematics of CNT types (A) M...

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Dyskusje

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 µm; MWCNT-OH: 8-15 nm x 50 µm; SWCNT: 1-2 nm x 1-3 µm. The powdered CNTs were dispersed in water by probe ultra-sonication. Aforementioned sizes of CNTs are likely to decr...

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Materiały

Name	Company	Catalog Number	Comments
Dimodan U	Danisco	15312	Store at 4 °C. Non-hazardous. Irritant to eyes and skin.
Phytantriol (> 95%, GC)	TCI Europe N.V.	P1674	Store at 4 °C. Non-hazardous. Irritant to eyes and skin.
Single walled Carbon Nanotubes (90%)	Nanostructured & Amorphous Materials, Inc.	1246YJS	Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system.
Multi-walled carboxylic acid functionalized Carbon Nanotubes (> 80% Caron basis, > 8% carboxylic acid functionalized)	Sigma-Aldrich Co. LLC	755125	Store at room temperature. Away from direct light. Causes serious eye irritation. May cause respiratory irritation.
Graphitized Multi-walled hydroxy functionalized Carbon Nanotubes (99.9%)	Nanostructured & Amorphous Materials, Inc. (NanoAmor)	1224YJF	Store at room temperature. Away from direct light. Irritating to eyes, skin and respiratory system.
Pluronic F127	Sigma-Aldrich Co. LLC	P2443	BioReagent, suitable for cell culture. Not a hazardous substance or mixture. Store at room temperature.
Acetone (99.5%)	Fisher Scientific	10134100	Highly flammable liquid. Causes serious eye irritation. May cause drowsiness or dizziness.
Jars with loose, enfolding lids (375 ml)	VWR International Ltd	216-3308
Beaker, 1,000 ml	Fisher Scientific	12942161	heavy duty, low form, with spout and graduations
Pasteur glass pipette (150 mm length) with latex bulb	Fisher Scientific	10006021
Microcentrifuge tube conical snap cap 1.5 ml	Fisher Scientific	11558232
Spatula	Fisher Scientific	11352204
Heating magnetic stirrer	Fisher Scientific	11715704
Magnetic stirrer bars (cylindrical, opaque PTFE, 30 mm x 7 mm (l x diameter))	Fisher Scientific	10011792
Needle (0.9 mm x 40 mm cannula length)	Terumo UK Ltd	MN-2038MQ
Retort Stand Set - With stand, clamp, base, rod, rubber 3 jaw and bosshead	Camlab Ltd, UK	1177157
Millipore water equipment	Barnstead Nanopure, Thermoscientific, USA
Progen Genfuge 24D Digital Microcentrifuge	Progen Scientific	C-2400
Probe ultra-sonicator, with 13 mm	SONICS, Vibracell,  USA
5 MP camera with auto-focus and LED flash	Samsung Galaxy Fame Mobile camera
Raman Spectrometer	Horiba Jobin-Yvon LabRAM HR800 spectrometer
Mastersizer 3000	Malvern Instruments Ltd, Malvern, United Kingdom
Small angle X-ray scattering (SAXS)	SAXSpace camera (Anton Paar, Graz, Austria), X-ray generating equipment (ISO-DEBYEFLEX3003, GE Inspection Technologies GmbH), closed water circuit (Chilly 35, HYFRA, Germany).