Nanoliposomal technology and food fortification aids each other. the bio availability of many compounds in food is low due due to their poor solubility or size or intolerance to gastric environment. nanoliposomes can be the answer to this.

1. Nanoliposomal Technology For Food &
Nutraceuticals
Bharti Sharma
MSc. Food Science & Technology (II Yr)

2. Food Nanotechnology
 The goal in food fortification is to ensure that the bioactive
compd.(s) are delivered and released at the proper time
and location in the body.
 The bioavailability of many compounds with poor water
solubility or low permeability in the small intestine can be
improved by appropriate control of food production and
consumption.
 Nanosize allow easier absorption after oral administration
as some particles can get absorbed by endocytosis and
these nanoparticles can easily move between cells of the
gastrointestinal tract .
 an increase in the surface area due to nanoparticles can
explain increased absorption
(Source: Rangan et al. 2016)

3. Types of Nano Based Delivery System
Type Size
range
Description Limitations
Nanoliposomes 10-300nm • Small vesicles surrounded by lipid bilayers
• Consists of phospholipids and cholesterol
• Physical instability
• Chemical degradation
Nanocochelates 50-500nm • Small vesicles surrounded by solid lipid bilayer
• Consist of phosphatidylserine, cholesterol and calcium ions
• Expensive
Micelles <100nm • Consist of surfactant molecules with a hydrophobic core
and a hydrophilic shell
• Large amount of surfactants
used
Nanoemulsions 10-100nm • Colloidal dispersion of two immiscible liquids (oil and
aqueous) with submicron size droplets
• Thermodynamically unstable
Cocervates 10-600nm • Biopolymer complexes of two oppositely charged protein
and/ or polysaccharides via electrostatic interactions
• Expensive
• Complex structure and
procedure
Source: Functional Foods and Dietary Supplements: Processing Effects and Health Benefits, Athapol Noomhorm, Imran

4. Nanoliposomes
 Bilayer lipid vesicles- hydrophilic head and
hydrophobic fatty acid tail
 Composed of phospholipids
 Are useful in areas of drug delivery, as diagnostic
agent and in food industries
 Nanoliposomes- nanometric version of liposomes.
 Liposome and nanoliposomes – similar in chemical,
structural and thermodynamic properties
 However, nanoliposomes provide more surface area
and have the potential to increase solubility, enhance
bioavailability, improve controlled release of the
encapsulated material to a greater extent.

5. Classification of Liposomes
On the basis of their size and number of bilayers, liposomes can also be classified as:
I. Multilamellar vesicles (MLV)- vesicles have an onion structure, making a
multilamellar structure of concentric phospholipid spheres separated by layers of
water
II. Unilamellar vesicles- the vesicle has a single phospholipid bilayer sphere enclosing
the aqueous solution.
Unilamellar vesicles can also be classified into two categories:
(1) large unilamellar vesicles (LUV >100nm)
(2) small unilamellar vesicles (SUV <100nm)

6. Method of Preparation
 Nanoliposomes are colloidal structures formed when phospholipid is mixed into an aqueous
solution using high energy input
 Input of energy (e.g. in the form of sonication, homogenisation, heating, etc.) results in the
arrangement of the lipid molecules, in the form of bilayer vesicles, to achieve a thermodynamic
equilibrium in the aqueous phase.
General methods of preparation
All the methods of preparing the liposomes involve four basic stages:
1. Drying down lipids from organic solvent.
2. Dispersing the lipid in aqueous media.
3. Purifying the resultant liposome.
4. Analyzing the final product.

7. Sonication
 Simple method for reducing the size of liposomes and manufacture of nanoliposomes.
 2 types of sonicators-
1. Probe Sonicator
2. Bath Sonicator
 For probe sonication, place the tip of the sonicator in the MLV flask and sonicate the sample for
10–15 min. At this stage, nanoliposomes are formed (SUV).
 Probe tip sonicators deliver high-energy input to the lipid suspension but suffer from
overheating of the lipid suspension causing degradation.
 Sonication tips also tend to release titanium particles into the lipid suspension, which must be
removed by centrifugation prior to use.

8. Bath Sonicator
• Fill the bath sonicator with room temperature water mixed with a couple of drops of
liquid detergent. Using a ring stand and test tube clamp, suspend the MLV flask in the
bath sonicator.
• The liquid level inside the flask should be equal to that of outside the flask. Sonicate for
20–40 min.
• Requires longer process times than probe sonication.
• Advantage- can be carried out in a closed container under nitrogen or argon, and does not
contaminate the lipid with metal from the probe tip.

9. Sonication
Dissolve lipids in
organic solvents
(chloroform)
Dry the mixture
using rotary
evaporator
Shake. Formation of
micrometric MLV
type liposome
Add suitable aqueous
solution to the dried
lipids
Remove traces of
organic solvent using
vacuum pump
Formation of
nanoliposomes
Transfer to
sonicator- bath-type
or probe (tip)
The aqueous medium can contain salts, chelating agents, stabilizers,
cryo-protectants (e.g. glycerol) and the drug to be entrapped. Source: Nanoliposomes: Preparation and Analysis-
M. R. Mozafari 2009

10. Extrusion Method
 Micrometric liposomes (e.g. MLV) are structurally modified to large unilamellar
vesicles (LUV) or nanoliposomes depending on the pore-size of the filters used.
 Vesicles are physically extruded under pressure through polycarbonate filters of
defined pore sizes.
 Preparation of sample such as MLV is done like before.
 Set up the extruder and place the polycarbonate filters on it.
 Load liposomal suspension in one syringe (donor) and place it in one end of the
extruder. Place the second syringe (receiver) in another end.
 Place the extruder stand onto a hot plate.

11. • Allow the temperature of the liposome suspension to reach the temperature of the
heating block (approximately 5–10 min).
• Gently push the plunger of the filled syringe until the liposome suspension is completely
transferred to the empty syringe.
• Gently push the plunger of the alternate syringe to transfer the suspension back to the
original syringe.
• Repeat the extrusion process for a minimum of seven passes through the filters.
• More passes though the filters, the more homogenous the sample becomes.
• Remove the filled syringe and inject the nanoliposome sample into a clean vial.
• When MLVs are forced through narrow-pore membrane filters under pressure,
membrane rupture and resealing occur and encapsulated content leaks out. Therefore,
extrusion is performed in the presence of medium containing the final drug
concentration, and external solute is removed only after formation is complete

12. Microfluidisation
 Method of production without using any potentially toxic solvent.
 Principle: dividing a pressure stream into two parts, passing each part through a fine orifice, and
directing the flows at each other inside the chamber of microfluidizer.
 Uses high pressures (up to l0,000 psi) to guide the flow stream through microchannels.
 Select the ingredients of the nanoliposomes and their suspension medium (an aqueous phase such
as deionized/distilled water or buffer) and mix them.
 Pass the dispersion through the microfluidizer. The process begins when the product enters the
inlet reservoir.

13. • An intensifier pump generates extremely high
pressures to accelerate the product to the interaction
chamber at velocities over 400 m/s. Inside this Y-type
chamber, the stream separates into microchannels,
which are as narrow as the cross-section of a human
hair.
• The suspension can be recycled through the
equipment in which case the suspension must be
cooled because of the temperature increase in the
interaction chamber at high operating pressure.
Disadvantage:
Material loss and the process involves a very high
shearing force that can potentially damage the structure
of material to be encapsulated.

14. Heating Method
 Method by which liposomes can be prepared using a single instrument without involving any toxic
solvent.
 Hydrate a suitable combination of the phospholipid components in an aqueous medium for a time
period of 1–2 h under an inert atmosphere such as nitrogen or argon.
 Mix the lipid dispersions along with the material to be encapsulated, in a heat-resistant flask such as
a pyrex beaker, and add glycerol to a final volume concentration of 3%.
 Place the flask on a hot-plate stirrer and mix the samples (800-1000rpm) until all lipids are
dissolved.

15. Depending on the nanoliposomal ingredients, sample volume, type of flask used and its
number of baffles, as well as type, speed and duration of mixing, nanometric vesicles can be
produced without the need to perform filtration or sonication.
The heating method has flexibility for the entrapment of various drugs and other bioactives
with respect to their temperature sensitivities, like-
• Adding the drug to the reaction medium along with the liposomal ingredients and glycerol;
• Adding the drug to the reaction medium when temperature has dropped to a point not
lower than the transition temperature (T c) of the lipids

16. Visualization of Nanoliposomes
 Phase Contrast microscopy- detect particles larger than 300 nm and contamination with larger
particles.
 Polarizing Microscope- reveal lamellarity of the liposomes
 Scanning Probe Microscopy- monolayers of various lipids and lipid attached molecules such as
antibody fragments can be studied

17. Involvement of Technology In Food
 Able to incorporate food antimicrobials that could aid in the protection of food products against
growth of spoilage and pathogenic microorganisms.
 One of the first reported liposome applications in food products was in cheese manufacturing.
The time and cost of cheese ripening can be reduced by the addition of proteinases to cheese
mixes before the isolation of curds. However, enzymes are often inactivated by the conditions of a
food system.
 The addition of free (i.e., unencapsulated) enzymes to the milk causes premature proteolysis,
resulting in unfavorable curd consistency and low yields.

18. A large proportion of the enzymes are lost in the whey stream, increasing product cost through
the requirement for a high initial enzyme concentration and limiting downstream whey processing
options.
 Moreover, the addition of the enzymes directly to curd results in poor enzyme distribution. It is
known that encapsulated enzymes have improved stability and activity not only in vivo, but also
during food processing in vitro.
 Liposomes isolate the encapsulated enzymes from the surrounding food environment, providing
them with protection under conditions that would otherwise impede activity or even cause
denaturation.
 Liposomes can also be used as a means of controlled release.

19. Advantages:
 sustained-release mechanism
 can encapsulate lipophilic and hydrophilic
material at the same time providing synergistic
effect.
 preserve desirable flavors and aromas or mask
unpleasant taste and appearance
 High bioavailability and absorption compared
with other oral forms of supplements.
 Micronized encapsulation protects against the
harsh environment of the GI tract
 Increased intracellular delivery.
 Ideal for those for whom swallowing a tablet is
not possible.
 Cost effective by being able to take a lower dose
for the same effect.
Disadvantages:
1. High current cost.
2. Possibility of poor manufacturing (eg,
high particle size, poor ingredients).
3. Possibility of instability.
4. Leakage of encapsulated material.

20. • Liposomes, and other lipid particles, are cleared from the blood by the reticulo-endothelial
system (RES), also called the mononuclear phagocyte system (MPS).
• Clearance is inversely related to size, with the small unilamellar liposomes/vesicles (SUVs)
and micronized emulsions circulating the blood the longest.
• Small liposomes may be drastically more efficient at intracellular delivery of encapsulated
compounds.
• In a recent study, with carefully-sized liposomes, cellular uptake increased 9-fold as liposome
size was decreased from 236 nm to 97 nm and was 34-fold higher at 64 nm.

21. • Modification of membrane surfaces with PEGs further enhances the ability of
liposomes and micronized emulsions to avoid detection by the cells of the RES,
thus protecting it from liver and spleen clearance and consequently prolonging its
circulatory time.
• Additionally, Stealth liposomes and micronized emulsions made by covering the
carrier surface with hydrophilic chains, such as PEG are more stable in
biological environments and can exhibit up to 10-fold higher circulation half-lives
than can liposomes without hydrophilic surface coatings.

22. • The FDA approved the first liposomal drug, a PEG-lated liposomal formulation of
doxorubicin for the treatment of Kaposi’s sarcoma in 1995.
• Among nutraceuticals, nanoliposomes suspensions of multichain fatty acids with
vitamin C that were prepared and freeze dried for long-term storage show
promising approach.
• Vitamin D3 was formulated as nanoliposomal product that can be used for
fortification of beverages.

23. INNOVATIVE DELIVERIES FOR NUTRACEUTICALS
 NutraLease™, Ltd. (Mishor Adumin, Israel)
developed a unique technology to produce micelles, which are self-assembled, structured liquid particles with a
diameter of 30 nm or less. These particles are designed to readily penetrate cell membranes and dramatically
increase the bioavailability of the phytonutrients carried and protected by the micelles .
 Proprietary Nutritionals Inc. (Kearny, New Jersey )
launched Benexia™ ALA Powder, neutral-tasting, water-soluble omega-3 microencapsulated powder for numerous
food applications
 LiveTheSource (Ft. Lauderdale, Florida)
announced its launch of “daily source,” the first ever nano-encapsulated liquid vitamin, mineral, and herbal
supplement. The company claims the herbal compounds in “daily source” will “provide a substantial increase in
nutritional value, first, due to their synergy, and second, due to their nano-encapsulation”.

24. References
 Nanotechnology In Food Products: Workshop Summary, Institute Of Medicine (US) Food Forum, Washington
(DC): National Academies Press (US); 2009.
 Liposome: classification, preparation, and applications
Abolfazl Akbarzadeh, Rogaie Rezaei-Sadabady, Soodabeh Davaran, Sang Woo Joo, Nosratollah Zargham, Younes
Hanifehpour, Mohammad Samiei, Mohammad Kouhi and Kazem Nejati-Koshki
 Nanoliposomes: Preparation and Analysis- M. R. Mozafari (2009)
 Nanostructured and nanoencapsulated natural antimicrobials for use in food products: A. Brandelli, T.M. Taylor
 Trends and Methods for nanobased delivery for nutraceuticals: Anupama Rangan, M.V. Manjula, K.G
Satyanarayana
 Nanodelivery of Nutrients for imporved bioavailability: Anu Bhushani, Udayakumar Harish, Chinnaswamy
Anandharamakrishnan 2017