Nonphospholipid Liposomes: Properties and Potential Use in Flavor

Chapter 17

Nonphospholipid Liposomes: Properties and Potential Use in Flavor Encapsulation Rajiv Mathur and Phil Capasso

Downloaded by FUDAN UNIV on March 11, 2017 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1995-0610.ch017

IGI, Inc., Wheat Road and Lincoln Avenue, Buena, NJ 08310

In recent years considerable effort has been directed toward the development of lipid bilayer vesicle delivery systems employing lipid amphiphiles other than phospholipids. Amphiphiles have been shown to form lipid vesicles of different types and stabilities. Our efforts have focused on the production of non-phospholipid paucilamellar vesicles that we called "Novasomes". These vesicles are 0.1-1.0 microns in diameter, with 2-5 bilayer shells surrounding an unstructured space that can be occupied by hydrophilic or hydrophobic materials. The vesicles can be produced in amounts ranging from milliliter continuous flow batches at costs equivalent to those of making simple emulsions. The uses of Novasome vesicles are extensive and range from cosmetics and pharmaceuticals, to foods. In all such applications, non-phoshpolipid Novasomes offer the advantages of high stability, low cost, and availability in large amounts. Novasome vesicles can be used for many functions in foods and beverages because of their ability to protect, transport, and deliver flavor oils, nutrients or other active ingredients.

Early work on lipid bilayer vesicles, as well as most current studies have dealt with structures madefromphospholipids. Since 1973, however, numerous experiments have shown that a lipid bilayer can be formed by molecules other than phospholipids. These molecules include fatty acids, dialkyldimethylammonium amphiphiles, dialkyl amphiphiles with ionic or zwitterionic head groups, polyglycerol alkyl ethers and polyethoxylated analogues, two-tailed sucrose fatty acid esters, and appropriate mixtures of single-tailed cationic and anionic surfactants (1-6). In 1987 scientists of Micro Vesicular Systems, Inc. (MVS) invented a new type of lipid microcapsule with unusual stability and transport properties called 0097-6156/95/0610-0219$12.00/0 © 1995 American Chemical Society Ho et al.; Flavor Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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Novasome lipid vesicle, hereafter named "Novasomes vesicles". These lipid microspheres are highly versatile and have been used in the encapsulation, transport, controlled release, and other manipulations of a wide array of molecules with applications forflavorand food, agriculture (7-8) and aquaculture, coating and paints (9), cosmetics and other personal care products, environmental cleanup and protection, pest control, animal and human biologicals and pharmaceuticals (JO-]]), in large amounts using MVS' patented, scalable continuousflowsystems {12-13). This review focuses on Novasome vesicles, uniquely structured, paucilamellar vesicles that can be formed from single-tailed non-phospholipid amphiphiles. They possess an aqueous or non aqueous core surrounded by one or several lipid bilayer shells, each composed of amphiphilic molecules, alone or in combination with a steroid (14-16). The manufacture of non-phospholipid liposomes, as here described, relies on the conversion of micellar solutions of membrane-mimetic amphiphiles to liposomal structures by manipulation of environmental variables (e.g., temperature, hydration) in an appropriate temporal sequence. As the concentration of a membrane-mimetic amphiphile in water increases, a sudden transition in the properties of the system occurs, representing the conversion of a molecular solution to a micellar solution. The transition concentration is known as the critical micelle concentration. The critical micelle concentration of the membrane-mimetic amphiphiles that we employ lie well below 10-°M. Micellar solutions consist of amphiphile molecules in small clusters (50-200 molecules) dispersed in water. The shapes of micellar clusters depends on the amphiphile type (e.g., head group, apolar chain) and the solution conditions (e.g., concentration, electrolytes, temperature). Micelle shape is determined by the surface area of the amphiphile molecules, the nature of the hydrophobic/hydrophilic interface and the curvature of that interface. Micelles form in various phases. Hexagonal phases consist of long, normal or reversed, rodshape structures with hexagonally packed arrays, and spherical micelles have been classified into two types of cubic packaging: body-or face-centered. The concentration of free amphiphile [A] is related to the proportion of amphiphile in micellar aggregates, [An] by a constant, K, which equals: [An]/[A]n = K

(1) n

When the concentration of free amphiphile [A] becomes equal to K"^ , equivalent to the critical micelle concentration, the formation of micelles begins and, as the value of [A] continues to increase above K " ^ , the concentration, [A ], of micellar aggregates rises rapidly. An increase in total amphiphile concentration above the critical micelle concentration thus implies an increase in the size and/or number of micelles, not the concentration offreeamphiphile. n

n

Ho et al.; Flavor Technology ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

17. MATHUR & CAPASSO

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Nonphospholipid Liposomes

The methods to be discussed for the manufacture of non-phospholipid liposomes involves the formation of micellar aggregates of membrane-mimetic amphiphiles and the controlled coalescence of these aggregates into membranebound vesicles (20). The membranes of such vesicles are stabilized by a number of interactions, including the hydrophobic effect driving apolar moieties out of water, van der Waals attractions between ordered amphiphile residues, and hydrogenbonding of amphiphile head groups to the water at the hydrophilic surfaces of each bilayer.


Major Constituents of Novasomes The non-phospholipid liposomes made by Micro Vesicular Systems, Inc. are engineered for particular applications. Appropriate Novasomes are assembled using a series of membrane "modules", each module imparting desired characteristics to the Novasome vesicles. The modular approach provides great breadth and flexibility to the membrane design (Figure 1). It also allows combination of numerous amphiphiles with each other, or with phospholipids to yield membrane hybrids with novel properties (21-24). Major Structure Modules. Table I lists some of the major structural membrane amphiphiles currently used in this non-phospholipid liposome vesicle designing for flavor and food application. In any given liposome type these molecules account for more than 50% of the membrane lipid. The fatty alcohols (Table I, #1) form bilayer vesicles only in association with a modulating module (Table II) and an ionogenic module.

Table I. Principal Wall-forming Material Classification

Hydrocarbon Chain

1. Fatty alcohol

Cn-Ca, (0-1 unsat.)

-OH

2. Fatty acid

Cu-Ca, (0-1 unsat.)

-COOH

3. Glycerol fatty acid monoester

Cu-Czo (0-1 unsat.)

-CO-0

-CH CHOHCH OH

4. Glycerol fatty acid diester

C -C C -C

(0-1 unsat.) (0-1 unsat.)

-CO-0 -CO-0

-CH-CHO

5. Propylene Glycol C^-C^ (0-1 unsat.) fatty acid monoester

-CO-0

-OCH CHOH CHj

12

12

lg

18

Bond

Head Group

2

2

2


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222

Q Q

•

Amphiphiles

Modulators

oo^oo

•

¥ ) O Q

Spacers

Ionogens

C O '

0

.00

A Novasome™ vesicle membrane bilayer Figure 1. bilayer.

Schematic representation of membrane modules of the Novasome




223


The fatty acids (Table I, #2) serve as structural modules for acid pHsensitive non-phospholipid liposomes. They form stable bilayers only when they are partly ionized (at bulk pH levels between 6 and 8, corresponding to membrane pH levels near 4-6) and in association with a nonionic amphiphile, such as fatty alcohol, and a modulator module (Table I). Full ionization of the carboxyl groups causes disruption of the bilayer, as does full protonation. Fatty acid monoesters and diesters (Table I, #3,4,5) form satisfactory lipid bilayer vesicles when appropriately combined with a modulator, spacer modules and an ionogenic module.

Table II. Membrane Modulators

Classification

Apolar Residue

Head Group

1. Cholesterol 2. Phytosterol (P)

A 5 Cholestene 3-P A 5 Stigmastene 3-P

-OH -OH

Modulator Modules. The modular approach used in the design of nonphospholipid liposomes relies on the use of sterol modulator molecules (Table II). Sterols can intercalate amphiphile hydrocarbon chains in bilayers, and thereby allow the intermixing of different acyl chains without phase segregation and broaden the range of temperature within which the crystalline- liquid-crystalline transition occurs. It appears that sterol interacts strongly with the polar termini of the chain, leaving them less free to change conformation than the more disordered long chain segments. Ionogenic Modules. The major ionogenic membrane amphiphiles currently used in our non-phospholipid liposomes which have GRAS status are fatty acids. They can be used in conjunction with any of the nonionic modules listed in Table I in preparations rangingfrom0.05 to 10 moles percent. Spacer Modules. Spacer modules are employed to widen the interlamellar space, reducing the number of lamellae, and to make the external bilayer less accessible than in the case with compact head groups. Polyoxyethylene derivatives of sorbitan fatty acid esters, fatty alcohols and fatty acids allow the placement atop the outermost bilayer surface of bulky residues acting as steric barriers to regulate interaction of the vesicle surface with, for example, viruses and phagocytitic cells.


FLAVOR TECHNOLOGY

224

1201

Addition of aqueous phase 100 \ Micelles ~~


P

I to-

Liquid

!I

4

*

18

I §

2

E

" (Turbulence)

t

il

So,ld

t

20 - Vesicles

k

0.00

o.so

1.00

Amphiphile (weight fraction in water)

120 too 80

1

60

2 Q. 40

E H-

20

o'— 0.00

Addition of aqueous phase Micelles emulsion droplets (Turbulence)

8

B

Liquid

I

Flavor oil

i

indifferent sulfactant

Oil core vesicles 0.50

Solid

1 .00

Amphiphile (weight fraction in water) Figure 2. Schematic representation of novasome vesicle formation. (A) Incorporation of aqueous medium only; (B) encapsulation of water and water immiscible fluid (oil, indifferent surfactant, etc.)




225

Chain Matching. Provided there are no steric or other interferences between head groups, the most stable membranes are produced by optimal matching of the chain length of the membrane modules. However, the action of sterols allows assembly of membranesfrommodules of varying chain length, thereby providing some control over permeability.


Preparation of Novasome Vesicles The manufacture of Novasome utilizes a unique process specifically designed for the formation of vesicles under rigorous conditions, in any amounts (from a few microliters to very large volumes), and at a cost equivalent to that of making simple emulsions. In laboratory experiments, two syringes, one containing the lipid and the other the aqueous phase, connected with a double hubbed needle are used. In the industrial process, vesicles are formed in a specially designed apparatus at volumeflowrates of 6 L/min and above. Vesicles with a Predominantly Aqueous Core. The generation of such vesicles is based on the known phase behavior of single-chain lipid amphiphiles at temperatures below 85°C. The membrane-forming amphiphile (plus modulating molecules such as sterol, fatty anions) is heated to give a liquid. This liquid is then injected at high velocity (10 to 50 m/s) through small channels (1 to 3 mm diameter) or needle (0.9 to 2.05 mm diameter) with turbulent mixing into excess aqueous phase and immediately cooled. The injection step causes the lipid to be disrupted into minute droplets that are quickly converted into micelles. Subsequent cooling under conditions of continued turbulence causes the micelles to fuse into paucilamellar vesicles within milliseconds (Figure 2A, Table III). This vesicle formation is facilitated and vesicle size distribution narrowed by inclusion of small amounts of nonamphiphilic, nonpolar, water-immiscible fluids in the lipid phase. Possibly microdroplets of these fluids segregate from amphiphile during micelle formation and subsequently act as nuclei around which micelles coalesce to form vesicles (Figure 2B). By use of this approach the loading of vesicles with water-soluble cargo can approximate theoretically 90% of the internal volume for vesicles 0.5 um in diameter, with two peripheral membranes. Vesicles with a Predominantly Water-immiscible Core (Flavor Oil). The preceding technique can be adapted for loading the novasome vesicle core with a wide variety of water-immiscible materials (including a broad range offlavoroils and nutrients) and solutions of substances in water-immiscible materials. Hot Loading. In this system, the water-immiscible, apolar substance is incorporated - together with a small amount of "indifferent surfactants" (surfactants that do not form bilayers) into the liquid lipid used to make novasome membranes. During injection of the liquid lipid into the aqueous phase, the water-


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FLAVOR TECHNOLOGY

immiscible substance forms microdroplets (diameter

Nonphospholipid Liposomes: Properties and Potential Use in Flavor

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