Technical Field

[0001] Colloidosomes are one type of microencapsulation whose shells are composed of colloidal particles. The

colloidosomes can be used to encapsulate a variety of active materials that may be slowly released over a specific period of time through the shells.

Brief Description of the Figures

[0002] The features and advantages of certain

embodiments will be more readily appreciated when considered in conjunction with the accompanying figures. The figures are not to be construed as limiting any of the preferred embodiments.

[0003] Fig. 1 is a schematic of a colloidosome having two different micro- or nano-materials making up an outer shell that contains an active material.

[0004] Fig. 2 is optical microscopy of multiple

colloidosomes .

[0005] Fig. 3 is optical microscopy of a colloidosome having a non-spherical shape.

Detailed Description of the Invention

[0006] Microencapsulation is the process of embedding or surrounding a core of active material within a shell.

Microencapsulation is commonly employed for materials such as flavors, fragrances, air fresheners, lotions, creams, nutrients, textile scents, drugs, etc., with goals such as: protecting active ingredients from degradation, improving material handling, and delaying or prolonging the release of the active material. Colloidosomes are one type of microencapsulation whose shells are composed of colloidal particles.

Nanostructured encapsulation systems (e.g., colloidosomes, encapsulated nanoparticles, liposomes, and the like) can provide improved uptake and efficient transport or delivery of active materials to intended targets (e.g., a person, an animal, an inanimate object, etc.) .

[ 0007 ] However, colloidosome systems have several limitations. By way of example, these systems tend to have a more spike or burst release profile of the active material rather than a more controlled and tunable release profile.

Furthermore, it is difficult to load multiple active materials into the core or in nested structures and thereby prevent having the active materials chemically react or mix with one another. Additionally, the loading capacity of the colloidosomes is limited by their core volumes, and the composition of the shells can impose thermodynamic limits on the type of active materials to be stored. Also, the types of stimuli that can be used to release active agents from the core are limited.

[ 0008 ] Because colloidosomes are microcapsules whose shells are composed of colloidal particles, there are inherent interstitial spaces between the colloidal particles in the shell. These interstitial spaces provide paths for leakage of active materials from the core. When the loaded active

material (s) are physically large such as antibodies or live cells (and physically larger than the size of the interstitial pores, ) leakage is less of a problem. However, when the active material is a molecule with an effective size the same or less than the interstitial spaces, for example, fragrances, then the leakage of active material (s) can be significant unless methods are used to hinder diffusion of the active materials (s) through the shell (e.g., sinter the colloidal particles into a contiguous shell, encapsulate the colloidosome in additional shell layers, or increase the length of the tortuous diffusion path) . Microencapsulation of fine fragrances presents a

formidable challenge, as the fragrance oils are relatively volatile, low molecular weight, and possess small effective sizes relative to the size of colloidosome interstitial pores. Additionally, the primary vehicle in fine fragrance is ethanol, which readily permeates/leaches across many shell materials.

[ 0009 ] Prior methods to control the leakage of active from colloidosomes include: multiple shell layers, sintering of the shell particles to form a contiguous shell, and over

encapsulation of the polymeric materials with a metal shell. Sintering of polymeric shell materials can be achieved, for example, by heating colloidosomes comprised of polystyrene styrene microgels to 105 °C. However, this method is not well- suited for active materials with relatively high volatility, because the active core will expand and burst the shell during heating .

[ 0010 ] Sintering presents material and temperature limitations, especially with volatile (low boiling point) active materials, while multiple layers add cost and complexity to processing, and can undesirably alter the release profile. In addition, sintering can result in uncontrolled fusion of shell colloidal particles that can impart characteristics of a single shell encapsulation and could be detrimental to the desired hindered diffusion phenomena that could be achieved in a

colloidosome architecture. Limited control over colloidal particle packing leads to the following problems in colloidosome microencapsulation: poor active encapsulation; high leak rate of active material from the colloidosomes; instability of the colloidosome architecture (with respect to time, temperature, or both) ; uncontrolled filling of the colloidosomes with active materials; disintegration of the colloidosome architecture under shrinking and swelling; and difficult or time-consuming active (re) filling of colloidosomes when multiple processing steps are employed after colloidosome formation or the colloidosomes are filled with active materials in a stepwise manner.

[0011] Thus, there is a need and an ongoing industry wide concern for improved colloidosomes that can be filled with an active material and released from the core in a more

controlled and sustainable manner.

[0012] It has been discovered that a colloidosome shell can be tunable to allow desired loading of one or more active materials into the cores of the colloidosomes. The tunable shells can also provide improved transport of the active

material from the core and through the shells and to the

intended recipient.

[0013] According to certain embodiments, a colloidosome comprises: a micro- or nano-structured porous shell comprising a plurality of micro- or nano-materials and interstices located between the micro- or nano-materials; and a core that is defined by the shell, wherein the shell is tunable to allow selective filling of an active material into the core and selective transport of the active material from the core through the shell. As used herein, the term "colloidosome" refers to a structure that has a shell defined by a plurality of micro- or nano-materials and interstices formed between the micro- or nano-materials and a core that is defined by the nanostructure shell .

[0014] The colloidosome includes a micro- or nano- structured porous shell comprising a plurality of micro- or nano-materials and interstices located between the micro- or nano-materials. The pore size of the micro- or nano-structured porous shell can vary depending on the application. Non- limiting examples of pore sizes range from about 0.5 nm to about 3 micrometers, about 10 nm to about 1,000 nm, or about 75 nm to about 200 nm, etc. In some embodiments, irrespective of pore size, the pore size can be tuned by surface functionality of the pore, where the surface functionalized molecule steric size can act as a pore blocking or gating tether.

[0015] The micro- or nano-structured porous shell can also include more than one type of micro- or nano-materials, for example, as shown in Fig. 1. The shell can include a first layer of the plurality of micro- or nano-materials. The first layer of the shell can define the core containing the active material. The shell can further include a second layer of a plurality of different micro- or nano-materials. The first plurality and the second plurality of the micro- or nano

materials can have the same or different geometries and the same or different particle sizes. According to certain embodiments, the shell can include a second micro- or nanostructured porous shell that encompasses the first shell and includes a second set of a plurality of micro- or nano-materials and interstices formed between the second set of micro- or nano-materials.

[0016] An overall size of the colloidosome can range from 5 to 30,000 nm, with the average size of the plurality of micro- or nano-materials being 2 nm to 15, 000 nm, and/or the size of the core being 5 nm to 2,500 nm, with the understanding that the size of the core is larger than the average size of the plurality of micro- or nano-materials that make up the shell.

The shape of the micro- or nano-materials can be a wire, a particle (e.g., having a substantially spherical shape), a rod, a tetrapod, a hyper-branched structure, a tube, a cube, a random structure, or mixtures thereof. As can be seen in Fig. 2, the colloidosome can have a substantially spherical shape. Fig . 3 depicts a colloidosome having a non-spherical shape.

[ 0017 ] The plurality of micro- or nano-materials can be microgel particles, nanogel particles, polymer brushes,

surfactant molecules, metal oxide particles, inorganic polymers, lipid particles, block copolymers, cross-linked polymers, biopolymers, biomolecules, micellular/dendrimer structures, yolk/shell structures, core/shell structures, pomegranate structures, hollow structures/nanomaterials, functionalized micro- or nanostructures, or combinations thereof. According to certain embodiments, , the plurality of micro- or nanomaterials are micro- or nanogel particles having a gel phase that include a polymer network of hydrophilic, hydrophobic, amphiphilic, amphiphobic, lipophilic, or lipophobic polymers, or a

combination thereof. The polymer network can be cross-linked.

[ 0018 ] The polymeric network can include non-ionic, cationic, anionic, or zwitterionic polymers or polymers that include metal-organic frameworks or zeolitic imidazolate

frameworks. Formation of the colloidosome can occur by

attaching the plurality of micro- or nanomaterials to one another. Such attachment can occur through a chemical bond, electrostatic interaction, van der Waals interaction, ionic interaction, hydrogen bonding, dipolar interaction, or any combination thereof. According to certain embodiments,

colloidosomes are formed by cross-linking microstructures, nanostructures, or both together. By way of example,

glutaraldehyde and/or 1 , 4-butanediol diglycidyl ether can be used to crosslink micro- or nanostructures formed from N¾- functionalized pNIPAAm. The micro- or nanostructured

architecture of the shell provides for superior mechanical properties (e.g., a yield strength of 1 kPa to 1 MPa or 1 kPa to 50 kPa) . [ 0019 ] A polymer is a large molecule composed of repeating units, typically connected by covalent chemical bonds. A polymer is formed from monomers. During the formation of the polymer, some chemical groups can be lost from each monomer.

The piece of the monomer that is incorporated into the polymer is known as the repeating unit or monomer residue. The backbone of the polymer is the continuous link between the monomer residues. The polymer can also contain functional groups connected to the backbone at various locations along the

backbone. Polymer nomenclature is generally based upon the type of monomer residues comprising the polymer. A polymer formed from one type of monomer residue is called a homopolymer. A copolymer is formed from two or more different types of monomer residues. The number of repeating units of a polymer is

referred to as the chain length of the polymer. The number of repeating units of a polymer can range from approximately 11 to greater than 10,000. In a copolymer, the repeating units from each of the monomer residues can be arranged in various manners along the polymer chain. For example, the repeating units can be random, alternating, periodic, or block. The conditions of the polymerization reaction can be adjusted to help control the average number of repeating units (the average chain length) of the polymer.

[0020] In a copolymer, the repeating units from each of the monomer residues can be arranged in various manners along the polymer chain. For example, the repeating units can be random, alternating, periodic, or block. As used herein, a "polymer" can include a cross-linked polymer. As used herein, a "cross link" or "cross linking" is a connection between two or more polymer molecules. A cross-link between two or more polymer molecules can be formed by a direct interaction between the polymer molecules, or conventionally, by using a cross- linking agent that reacts with the polymer molecules to link the polymer molecules.

[ 0021 ] The polymers can be selected from the group consisting of poly (N-isopropylacrylamide) (pNiPAAm), N- isopropylacrylamide - (N, N' -methylenebisacrylamide ) copolymer, N-isopropylacrylamide - (N, N' -methylenebisacrylamide) -allyl amine copolymer ("Nfh-functionalized pNiPAAm", ) polyethylene glycol, functionalized pNiPAAm, polyvinyl alcohol (PVA) , hydroxylated poly (meth) acrylate, ethylene-vinyl acetate

copolymer, 2-hydroxyethyl methacrylate (HEMA) , polymaleic acid/octyl vinyl ether (PMAOVE) , polyurethane, poly (acrylic acid), poly(stearyl acrylate) (PSA), poly ( acrylamide ) ,

polyolefin, poly lactic acid (PLA) , polylactic-coglycolic acid copolymer (PLGA) , alginate, chitosan, polyesters (eg

poly (pentadecanolide ) , polyethylene terephthalate (PET), polybutylene terephthalate (PBT) , poly (butylsuccinate ) ) , polyester copolymers (e.g., lactic acid/lysine copolymer), glycerol copolymers, polycarbonates, polyetherimides ,

polyphenyloxides , polystyrenes, poly (methyl methacrylate)

(PMMA) , copolymers of any of the foregoing, and combinations thereof .

[ 0022 ] The colloidosome can be formed by the addition of a cross-linking agent to form the micro- or nano-structured porous shell. By way of example, poly lactic acid is a polymer that does not need to be cross-linked; however, an example of a polymer that can be cross-linked is bis-acrylamide cross-linked pNiPAAm. A cross-linking agent can be added to help stabilize the colloidosomes formed from polymeric micro- or nano

materials .

[ 0023 ] The core of the micro- or nano-structured porous shell includes an active material. There can also be more than one type of active material in the core. The core of the colloidosome can be a polymer emulsion, a polymer gel, an aerogel, a liquid (e.g. an oil), or a partially void space. The active material can be any active material that is desirably released from the core in a desired period of time. Examples of active materials include, but are not limited to, a chemical agent, a biological agent, an oil, an ionic liquid, a

suspension, a polymer, and combinations thereof. Chemical agents can include a drug, gaseous molecules, a cosmetic agent, a flavoring agent, a fragrance-producing chemical, a malodor agent, a reactive agent, a cross-linker, a reactive diluent, a solvent, an inorganic or organic chemical, a organometallic system, a petrochemical, a reducing or oxidizing agent, or an aqueous salt, or any combination thereof. The biological agent can include a protein, a peptide, a nucleic acid, a

carbohydrate, a lipid, or any combination thereof. Non-limiting examples of such compositions include pharmaceutical

compositions, cosmetic compositions, personal care products, fragrances, perfumes, compositions intended for inanimate objects or surfaces (e.g., cleansers, disinfectants, dish detergents, laundry detergents), etc.

[0024] The colloidosomes can be used in a variety of applications ranging from drug delivery, catalysis,

nanocomposites, bioanalysis, diagnostics, sensors and markers, energy storage, bio-inhibitors (repellants pesticides,

herbicides), urea release, self-repair (e.g., paints, paper, textile, concrete, etc.), flame retardancy, personal care (e.g., skin care, fragrances or perfumes, hair, teeth, etc. ) ,

nutritional additives, vitamins, flavors, pigments, textile scent, detergents, softeners, animal care, lubricants,

adhesives, etc.

[0025] According to certain embodiments, the interstice sizes of the colloidosome are substantially uniform. That is, at least about 90%, or about 95%, or even about 100% of the interstices of the colloidosome can be about the same size.

These interstices can, for example, have the same average diameter or vary no more than about 10%, about 5%, or about 2% of the average diameter. The average diameter of a non-circular interstice is the diameter of a circle having the same surface area as that of the interstice. In other embodiments, the radius of the interstice may differ by about 50% to about 300%, resulting in interstice differing in diameter by up to a factor of about 1.5, or even by a factor up to about 4. In yet another embodiment, the interstice may differ in radius by up to about 50%.

[ 0026 ] The shell is tunable to allow selective filling of an active material into the core and selective transport of the active material from the core through the shell. The active material is transportable from the core through the micro- or nano-structured porous shell. Transport of the active material can be achieved through diffusion or release. The shell can also be tunable to provide an optimum release (i.e., premature release of the active material is substantially inhibited or prevented) of the active material from the core.

[ 0027 ] According to certain embodiments, the shell is tunable by altering the normalized packing density of the micro- or nano-materials. The normalized particle density can be in the range from about 0.5 to about 1.2. The normalized particle density p-d _n ² is used here to characterize the colloidosome structure and obtain a relation with the number-average particle diameter d _n . The density p is defined as the number of particles per unit area, which is obviously different for each particle size. Therefore, it has to be normalized by multiplying with the square of the diameter in order to make a fair comparison among the different particle sizes. [ 0028 ] The normalized packing density can be altered via changes to: an interaction between individual micro- or nano particles (e.g., altering the dielectric constant, short range forces, long range forces, hydrophobic interactions, and ionic interactions); inter-particle cross-linking; colloidosome morphology size and shape; reactivity and functionality of a cross-linker; extent of cross-linking (e.g., cross-linker concentration, number of cross-linking treatments, density of functional sites on the surface of the micro- or nano-materials, cross-linker reactivity, and temperature) ; or morphology of the micro- or nano-particles.

[ 0029 ] The use of a cross-linking agent can be used to tune the micro- or nano-structured porous shell to achieve desired properties. By way of example, during the formation of the colloidosome, the use of either multiple and/or different (with regards to length, functionality or both) cross-linking agents or multiple rounds of treatments with the same cross linker can provide tight colloidal particle packing enabling reduced leakage. As an example, when an active material is loaded after the step of synthesizing the colloidosomes , it can be advantageous to retain porosity in the shell. A porous shell allows ingress or loading of the active material, which can subsequently be locked in place by techniques such as adding additional cross-linking agents (with the same or different cross-linkers) or by adding one or more shells. Such an

approach is desired for commercial ease of use where a customer fills the "empty" colloidosomes with a proprietary mixture

(e.g., fine perfume) . Once the customer fills the porous colloidosomes with the active material, a second reagent that reduces the porosity and leak rate can be added. With such an approach, the empty, porous colloidosomes can be provided as a product and encapsulation is performed by the customer at his/her premises.

[ 0030 ] According to certain other embodiments, cross linkers that are responsive/reactive to changes in reaction conditions can be utilized. For example, the cross-linking rate and density can be modulated by altering: the solution medium, pH, the polarity of the solution, temperature, pressure, etc.

[ 0031 ] According to certain other embodiments, cross linkers can be used that possess physiochemical properties that affect active transport across the shell, adjusting the

interaction with the bulk medium in which colloidosomes are dispersed, or directing the assembly of additional shells over the colloidosome . An example is loading a colloidosome with a lipophilic oil, then performing cross-linking with cross-linker molecules that possess hydrophilic or ionically-charged regions between reactive groups, thereby providing an energetic barrier to diffusion across the shell. A cross-linker with an

ionically-charged "spacer" portion between the two or more reactive ends can be used to create a composite electrostatic barrier comprised of both an ionically charged medium around the colloidosome and ionically charged cross-linkers. The choice of cross-linker can in this way change the transport/barrier properties of the shell. Additionally, the cross-linker

functionality can be used to influence the interaction of the architecture with the surrounding medium (e.g., fragrance formulation) and lead to a partially or fully stable dispersion.

[ 0032 ] According to certain other embodiments, the shell can be tunable by using cross-linkers that can be isomerized, and thereby changing the normalized packing density upon

isomerization. One example is cross-linkers possessing

asymmetric alkenes. Ultraviolet light can be used to induce a trans-cis conversion, effectively shortening the cross-linker length and bringing neighboring colloidal shell particles closer together, and thus, changing the normalized packing density.

[0033] The cross-linker can be selected from the group consisting of bis isocyanates (e.g., lysine diisocyanate ethyl ester), bis epoxides (eg, 1 , 4-butanediol diglycidyl ether,) activated esters (e.g., N-hydroxysuccinimide esters,

pentafluorophenyl esters,) bis or poly amines (e.g., 1,6- diaminohexane, spermidine) , bis lactones, acid anhydrides, bis carboxylic acids, and combinations thereof. The concentration of the cross-linker can vary and can be selected to tune the normalized packing density of the micro- or nano-materials.

According to certain embodiments, the cross-linker is in a concentration in the range of about 0.5 mM to about 5 M.

[0034] It is to be understood that one or more different cross-linkers can be used to tune the micro- or nano-structured porous shell by adjust the normalized packing density. In some embodiments, more than one cross-linker is used to form the colloidosome with the cross-linkers being the same (but with different concentrations) or different. The two or more cross linkers can be added at the same time or at different times during or after manipulation or treatment of the colloidosome.

[0035] The shell can also be tunable by adjusting the morphology (i.e., the size and shape) of the micro- or nano materials forming the shell. By way of example, the physical size of the colloidal particles can be selected or controlled. The micro- or nano-materials can have different sizes and the shapes of the shell can be altered to provide shells having round, oval, elliptical, etc. shapes. Examples include: using polydispersity in nano- or micro-particle sizes to enable use of non-uniform size distribution in order to achieve packed shells of colloidosomes and thus, eliminate time-consuming and/or expensive approaches to obtain narrow distribution or size control of microgels. Such colloidosomes may significantly reduce thermodynamic phenomena such as the Ostwald ripening effect that can cause destabilization of the colloidosome architecture wherein the encapsulated active materials can prematurely leak out. The approach of having polydisperse microgels also allows for minimizing the dynamic changes in colloidosome sizes in a specific formulation. This can be achieved in single or multi-shell architectures.

[ 0036 ] The morphology can also be adjusted by selecting or controlling the geometry of the colloidal particles

comprising the colloidosome shell (s.) Examples include:

different or the same sizes and shapes of the micro- or nano materials; combining swollen or shrunken micro- or nano

materials; micro- or nano-materials with the same or different multiple chemical functionalities to tune the cross-linking and tight packing of shell (s); and enabling tight packing by

electrostatics.

[ 0037 ] The shell can be tunable by altering the

dielectric constant of the colloidosome ' s surroundings. The dielectric constant can be altered by the addition of

dissociative additives (e.g., salts such as LiCl, polyanions, or polycations such as spermidine in water at neutral pH) to change the dielectric constant of the surface of the micro- or nano materials or the medium around the colloidosome, thereby

creating an energetic barrier to diffusion of uncharged

molecules across the colloidosome shell. Charges from particles or moieties can be used to force the composition of the

colloidosome to either stay intact, or release the active material at a rate controlled by the charge type/density.

[ 0038 ] The core of the colloidosome can be re-filled after transport of the active material from the core through the shell. According to certain embodiments, after transport of the active material through the shell, the shell remains

substantially intact; thereby allowing a second active material to be loaded into the core. According to certain other

embodiments, the cross-linking bonds are reversible and the shell can be refilled with the same or different active material after transport of the active material through the shell. This can be achieved by using cross-linkers that are chemically reversible. One example of a reversible cross-linking bond is thiol-disulfide bonds. If a cross-linker possesses a disulfide bond, the bond can be readily cleaved to thiols with a reducing agent, thereby lowering the packing density, and later re oxidize to the disulfide, thereby restoring the normalized packing density.

[0039] Methods of producing the colloidosome can include combining micro- or nano-materials, a base fluid, and an active material to form a first fluid; adding a cross-linking agent to the first fluid to form a second fluid; and allowing the cross- linking agent to cross-link the micro- or nano-materials to form a micro- or nano-structured porous shell comprising a plurality of the micro- or nano-materials and interstices located between the micro- or nano-materials, and wherein the active material is located within a core that is defined by the shell.

[0040] The base fluid can include water, a liquid hydrocarbon, a solvent (e.g., benzene, hexane, xylene, methanol, ethanol, dichloromethane, etc.), or mineral oil, edible oils (e.g., sunflower oil), toluene, or silicone fluids.

[0041] The method can further include adding a second micro- or nano-material to the second fluid. As discussed above, the methods can further include more than one addition of the cross-linking agent to form the second fluid, for example, utilizing multiple additions of the cross-linking agent over a specified period of time. [0042] According to certain other embodiments, a method of making a colloidosome can include: combining micro- or nano materials, a solvent, and an active material to form a first fluid; adding a first cross-linking agent to the first fluid to form a second fluid; adding a second cross-linking agent to the second fluid; and allowing the cross-linking agent to cross-link the micro- or nano-materials to form a micro- or nano-structured porous shell comprising a plurality of the micro- or nano materials and interstices located between the micro- or nano materials, and wherein the active material is located within a core that is defined by the shell. The first cross-linking agent and the second cross-linking agent can be different. This method can also include adding a second micro- or nano-material to the second solution before, during, or after the addition of the second cross-linking agent.

[0043] A method for altering the dielectric constant can include: dispersing micro- or nano-materials in a working fluid; creating an emulsion by combining the working fluid with a liquid hydrocarbon; and adjusting the ionic strength of the emulsion with an electrolyte. The electrolyte can be selected from the group consisting of salts (e.g., sodium chloride, potassium chloride, calcium chloride, etc.) in aqueous

solutions, salts in organic solutions, ionic liquids, and combinations thereof. This method can further include adding a second micro- or nano-material to the emulsion before the step of adjusting the ionic strength. According to certain

embodiments, a surfactant or emulsifier can be added to the working base fluid and the liquid hydrocarbon to create the emulsion. The surfactant or emulsifier can be selected from ionic, anionic, non-ionic, cationic, zwitterionic, or neutral surfactants, and combinations thereof. Examples

[0044] To facilitate a better understanding of the present invention, the following examples of certain aspects of preferred embodiments are given. The following examples are not the only examples that could be given according to the present invention and are not intended to limit the scope of the

invention .

[0045] The following example illustrates a method of preparing colloidosomes with one type of cross-linking agent and one type of micro- or nano-material (microgel) :

Example 1 : 50 milligrams (mg) of amino-functionalized poly(N- isopropylacrylamide ) (pNiPAAm) microgels (lyophilized NH ₂ - pNiPAAm) having a diameter of approximately 0.5 to 1.1 micrometers (pm) (500 to 1,100 nanometers (nm) ) were dispersed in 5 grams (g) of water with vigorous stirring. 0.6 g of an active material of limonene was then added. Stirring was continued overnight. 84 mg of a cross-linking agent of 1,4- butanediol diglycidyl ether was then added in portions.

Stirring was continued at room temperature.

[0046] The following example illustrates a method of preparing colloidosomes with one type of cross-linking agent and one type of micro- or nano-material (microgel) added at two different stages:

Example 2 : 26 mg of polylactic-coglycolic acid copolymer (PLGA) (50:50 L:G monomer ratio, 15-25 kDa) microgels (lyophilized solid) having a diameter of approximately 200 to 300 nm were added to 2.5 g of 10 mM phosphate buffer, pH 7, in a glass scintilliation vial with stirbar. The mixture was stirred at 1,000 revolutions per minute (rpm) for approximately 5 min. 129 mg of an active material of limonene was then added and the stir rate was increased to 1,200 rpm for 10 min. Stirring was

discontinued, and 5 mg of a coupling agent, N-3- dimethylaminopropyl ) -N' -ethylcarbodiimide hydrochloride (EDC-HC1) was then added to the reaction, and the sample was placed on rotary orbital shaker at 150 rpm. After 3 min, 22 mg of N¾-capped PLGA (50:50 L:G monomer ratio, 10-15 kDa)

(lyophilized solid) having an approximate diameter of 300 nm were added. The reaction was swirled to mix and placed on the rotary orbital shaker at room temperature.

[ 0047 ] The following example illustrates a method of preparing colloidosomes with one type of cross-linking agent and two different types of micro- or nano-material (microgels) :

Example 3 : After 2 days, a 1 g portion of the reaction mixture from Example 1 was removed and combined with 2 g of water and approximately 10 mg of a second type of pNiPAAm microgels, carboxylate-functionalized microgels (CCpH-pNIPAM) having a diameter of 0.2 to 0.5 pm (200 to 500 nm) and shaken on a rotary orbital shaker to produce a mixed or layered NH ₂ -pNIPAM and CCpH- pNIPAM colloidosome shell surrounding the limonene core.

[ 0048 ] The following example illustrates a method of preparing colloidosomes with one type of cross-linking agent at multiple additions and one type of micro- or nano-material

(microgel) :

Example 4 : 50 mg of reconstituted lyophilized Nf^-functionalized pNiPAAm microgels having an approximate diameter of 0.5 to 1.0 pm were dispersed in 5 g of water. 0.6 g of an active material of limonene was then added with vigorous stirring. 84 mg of a cross-linking agent of 1 , 4-Butanediol diglycidyl ether (20 times reactive equivalents) was then added in four different time frames over a period of approximately 8 h. The solution was aged at room temperature with stirring to form colloidosomes with a cross-linked pNiPAAm polymer shell surrounding the

limonene core. [0049] The following example illustrates a method of preparing colloidosomes with two different types of cross- linking agents and two different types of micro- or nano materials (microgels) :

Example 5: A 1 g portion of the intermediate reaction product (cross-linked amino-pNiPAm colloidosomes) listed in Example 1 was combined with 10 mg of a second type of microgel of

carboxylate-functionalized microgels (pNiPAM in 2 g water, CCpH- pNilPAAm) having a diameter of 0.2 to 0.5 pm (200 to 500 nm) .

22 mg of a second type of cross-linking agent of N-3- dimethylaminopropyl ) -N' -ethylcarbodiimide hydrochloride

(EDC-HC1) was then added, and the mixture was shaken at room temperature on a rotary orbital shaker to produce a mixed NH ₂ - pNiPAAm and CCpH-pNiPAAm colloidosomes with a limonene core and covalent amide cross-links between the microgels.

[0050] The following example illustrates a method of preparing a colloidosome by altering the dielectric constant of the micro- or nano-material shell:

Example 6: Poly (lactic acid) (PLA) microgels having a mean diameter of approximately 200 nm were dispersed in 5 milliliters of water at a concentration of 20 mg/mL. While stirring at

1,200 rpm, 0.2 g of an active material of limonene was added slowly to the microgel dispersion, and the reaction was mixed for 10 min. 10 mg of formulation aid DC5200 available from Dow Chemical, U.S.A. was then added to the solution and the mixture was emulsified with probe sonication for 10 s. The resulting emulsion was then added drop-wise through a 26-gauge needle to a second 10 mL dispersion of PLA microgels (2 mg/mL) while

stirring at 500 rpm for 1 h. Lastly, 100 microliters of a 1,000 ppm sodium chloride solution electrolyte was added to the

reaction and the mixture was stirred for 0.25 h. [0051] The following examples test the releasability of an active material from the core for different colloidosomes .

[0052] The colloidosome for the testing results shown in Fig. 4 was prepared in a vial by mixing 1 mL of pNIPAM-NH ₂ nanogels (concentration 10 mg/mL) , either 1 or 10 mL of

glutaraldehyde (25%), 40 mg Dowsil 5200, 1 mL of limonene

(99.5%), and 10 mL of DI water. The sample was then sonicated using a probe sonicator at 50% intensity for 30 seconds on ice. The solution was then heated at 60 °C for 30 minutes. After heating, the sample was then aged at room temperature for 72 hours. Tenacity testing samples were prepared after blotting 20 mg of colloidosomes onto a filter paper. Tenacity was measured by a GC headspace method. As can be seen in Fig. 4, the heavier cross-linked (10 mL GA) sample released less active material (limonene) when compared to the lightly cross-linked (1 mL GA) sample. This shows that the amount of active material released can be controlled by tuning the shell to provide optimum release of the active core.

[0053] The colloidosomes for the testing results shown in Fig. 5 were prepared in a vial by mixing the following ingredients :

Samp1e A

1 mL of pNIPAM-NH ₂ nanogels (concentration 10 mg/mL) ;

2 mL of glutaraldehyde (25%);

46.7 mg of Dowsil 5200;

1 mL of limonene (99.5%); and

10 mL of DI water.

Samp1e B

0.5 mL of pNIPAM-NH ₂ nanogels (concentration 10 mg/mL);

0.5 ml of pNIPAM-C0 ₂ H nanogels (concentration 10 mg/mL);

2 mL of glutaraldehyde (25%);

44.6 mg of Dowsil 5200; 1 mL of limonene (99.5%); and

10 mL of DI water.

The samples were then sonicated using a probe sonicator at 50% intensity for 30 seconds on ice. The solutions were then heated at 60 °C for 30 minutes. After heating, the samples were then aged at room temperature for 72 hours. Tenacity testing samples were prepared after blotting 20 mg of colloidosomes onto a filter paper. Tenacity was measured by a GC headspace method.

As can be seen in Fig . 5 , Sample B had a slower release of the active material (limonene) due to the greater packing density of neighboring nanogels terminated with different chemical end groups (i.e., amine and carboxylic acid groups) . This shows that the release time of the active material can be controlled by changing the interaction between particles, inter-particle cross-linking, colloidosome morphology, reactivity or

functionality of a cross-linker, extent of cross-linking, and/or morphology of colloidal particles.

[ 0054 ] The following test shown in Fig . 6 was performed to show re-loading of an active material into the colloidisomal shell. The test was performed by preparing a colloidosome by mixing 1 mL of rNIRAM-N¾ nanogels (concentration 10 mg/mL) , 10 mL of glutaraldehyde (25%), 40 mg Dowsil 5200, 1 mL of limonene (99.5%), and 10 mL of DI water. The sample was then sonicated using a probe sonicator at 50% intensity for 30 seconds on ice. The solution was then heated at 60 °C for 30 minutes. After heating, the sample was then aged at room temperature for 72 hours. The active material limonene was removed from the colloidosomes by immersing the colloidosomes in a IPA solution overnight. The collected precipitate was re-dispersed into a guanine solution to re-load the core with guanine. Excess guanine was removed by centrifuge. A control sample of guanine was prepared without encapsulation. Tenacity testing of the samples was prepared after blotting 20 mg of colloidosomes onto a filter paper. Tenacity was measured by a GC headspace method. The results in Fig . 6 show that guanine was re-loaded into the colloidosome shell compared to the control without

encapsulation. This demonstrates that the shell remains

substantially intact after release of a first active material and can be re-loaded with a second active material for

subsequent release.

[ 0055 ] The colloidosomes for the testing results shown in Fig . 7 were prepared in a vial by mixing the following ingredients :

Samp1e A

1 mL of pNIPAM-NH ₂ nanogels (concentration 10 mg/mL) ;

10 mL of glutaraldehyde (25%);

40 mg of Dowsil 5200;

1 mL of limonene (99.5%); and

10 mL of DI water.

Samp1e S

1 mL of pNIPAM-NH ₂ nanogels (concentration 10 mg/mL) ;

10 mL of glutaraldehyde (25%);

40 mg of Dowsil 5200;

1 mL of limonene (99.5%);

10 mL of DI water; and

0.0149 g NaCl (50 mM) .

The samples were then sonicated using a probe sonicator at 50% intensity for 30 seconds on ice. The solutions were then heated at 60 °C for 30 minutes. After heating, the samples were then aged at room temperature for 72 hours. Tenacity testing samples were prepared after blotting 20 mg of colloidosomes onto a filter paper. Tenacity was measured by a GC headspace method.

As can be seen in Fig . 7 , sample S has a slower release of the active material (limonene) due to the adjustment of the ionic strength by adding an electrolyte (NaCl) . This shows that the release time of the active material can be controlled by

adjusting the ionic strength of the solution with the addition of an electrolyte.

[0056] Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is, therefore, evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention.

[0057] As used herein, the words "comprise," "have," "include," and all grammatical variations thereof are each intended to have an open, non-limiting meaning that does not exclude additional elements or steps. While compositions, systems, and methods are described in terms of "comprising," "containing," or "including" various components or steps, the compositions, systems, and methods also can "consist essentially of" or "consist of" the various components and steps. It should also be understood that, as used herein, "first," "second," and "third, " are assigned arbitrarily and are merely intended to differentiate between two or more particles, shell layers, etc., as the case may be, and does not indicate any sequence.

Furthermore, it is to be understood that the mere use of the word "first" does not require that there be any "second," and the mere use of the word "second" does not require that there be any "third," etc. [ 0058 ] Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In

particular, every range of values (of the form, "from about a to about b, " or, equivalently, "from approximately a to b, " or, equivalently, "from approximately a - b") disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles "a" or "an," as used in the claims, are defined herein to mean one or more than one of the element that it introduces. If there is any conflict in the usages of a word or term in this specification and one or more patent (s) or other documents that may be incorporated herein by reference, the definitions that are consistent with this specification should be adopted.