WO2013015674A1 - Collagen nanostructures - Google Patents

Abstract

The present invention relates to a collagen nanostructure of diameter less than 300nm, formulation comprising the same, methods for the production of the same, as well uses thereof.

Description

COLLAGEN NANOSTRUCTURES

FIELD OF THE INVENTION

The present invention relates to a collagen nanostructure, formulations comprising the same, methods for production of the same, as well as uses thereof. In particular, the invention relates to collagen nanostructures such as nanoparticles, liposomes, micelles and/or niosomes.

BACKGROUND TO THE INVENTION

In recent years, there have been exciting developments in tissue engineering and wound healing. Much of these advancements utilize collagen as a substrate as collagen has many attractive properties that allow it to be used in various medical applications, such as implants, transplants, organ replacement, tissue equivalents, vitreous replacements, plastic and cosmetic surgery, surgical suture, surgical dressings for wounds, burns, and the like. Collagen is the principal component of connecting tissues of mammals. By itself, it is a relatively weak immunogen, at least partially due to masking of potential antigenic determinants within the collagen structure. It is also resistant to proteolysis due to its helical structure. Collagen is a natural substance for cell adhesion and the major tensile load-bearing component of the musculoskeletal system. Thus, extensive efforts have been devoted to the production of collagen fibres and membranes suitable for use in medical, as well as veterinary applications.

Collagen preparations for use in various medical applications are typically prepared from skin, tendons (e.g., bovine Achilles, tail, and extensor tendons), hide or other animal parts, by procedures involving acid and/or enzyme extraction. Methods for collagen preparation usually involve purification of collagen by extraction with diluted organic acids, precipitation with salts, optional gelation and/or lyophilization, tangential filtration and the like. An example of the method includes first separating fascia, fat and impurities from tissue. The tissue is then subjected to moderate digestion with proteolytic enzymes, such as pepsin. The collagen is then precipitated at a neutral pH, redissolved and the residual impurities precipitated at an acid pH. The resultant tissue is then digested with a strong alkali and then exposed to acid to facilitate swelling. The collagen fibres are then precipitated with salts or organic solvents, and dehydrated as disclosed in US patent no. 5,028,695. The extracted collagen can further be converted into a finely divided fibrous collagen by treating water-wet collagen with acetone to remove water, centrifuging to obtain the solid mass of collagen and disaggregating the collagen during drying as provided in U.S. Pat. No. 4,148,664. The collagen preparation can then be brought back to a neutral pH and dried in the form of fibres. Completely transparent, physiological and hemocompatible gels, collagen films, and solutions can be prepared from the collagen resultant preparation. These forms of collagen may then be used in the fabrication of contact lenses and implants.

However, while naturally occurring collagen has been increasingly used in the medical, veterinary and cosmetic fields, its relatively large molecular size has precluded it from much broader use. For example, with the fundamental structural unit of 5 collagen being a molecular rod about 2600 A in length and 15 A in diameter with a molecular weight of 300,000 Daltons, its ability to penetrate skin and the like is limited. While there are roles for collagen in tissue engineering that requires the breakdown and reabsorption to be relatively lengthy, there are other uses for collagen where the relatively rapid reabsorption is of interest. This cannot be achieved by the collagen obtained from the methods of collagen preparation known in the art.

Also, in the topical use of compounds such as collagen the penetration of collagen into skin is a major problem. The penetration is linked to the degree of permeability of the skin (which is linked to its physiological condition) and to the physicochemical properties of the compounds which need to enter the skin. For example, molecular weight, polarity, ionization stage and the like of the compound together with the nature of the environment (excipient, carrier and the like used in the compound together) affect whether the compound is going to penetrate the skin.

The skin serves numerous functions, but its primary function is as a protective layer or barrier. The most important role of the skin for terrestrial animals is to protect the water- rich internal organs from the dry environment. This cutaneous barrier function of the skin resides in the upper most thin layer (approximately 10-20 pm in humans) called stratum corneum. The water impermeability of this layer is 1000 times-higher than that of other membranes of living organisms. SUMMARY OF THE INVENTION

Accordingly, in order to open up new areas for the use of collagen, especially in the treatment of skin disorders or where collagen might be used as a carrier for the topical administration of chemical compounds or drugs through the skin there is a need for an improved form of collagen.

In a first aspect, the present invention provides a collagen nanostructure with a diameter less than 300nm. The nanostructure may be a nanoparticle, liposome, micelle and/or noisome. In particular, the nanostructure may be capable of enhanced penetration and/or retention effect in at least one cell relative to a control.

According to another aspect, the present invention provides a collagen nanostructure formulation comprising a plurality of nanostructures according to any aspect of the present invention. In particular, at least 70% of the nanostructures in the formulation have a diameter less than 250nm.

According to a further aspect, the present invention provides a method of producing a collagen nanostructure and/or a collagen nanostructure formulation according to any aspect of the present invention, the method comprising the steps of:

(i) reducing at least one metal ion with at least one reducing agent;

(ii) adding at least one polymer to (i);

(iii) adding at least one collagen solution to (ii);

(iv) adding at least one cross-linker to (iii).

According to other aspects, the present invention provides a collagen nanostructure and/or collagen nanostructure formulation prepared according to the method of the present invention, a method of treating a disease, in particular a skin disease by administering the collagen nanostructure and/or formulation according to any aspect of the present invention and/or the collagen nanostructure and/or formulation according to any aspect of the present invention for use in treating a disease, use of the collagen nanostructure and/or formulation according to any aspect of the present invention for the preparation of a medicament and uses thereof. As will be apparent from the following description, preferred embodiments of the present invention allow an optimal use of the collagen nanostructures to take advantage of their size, penetration and/or retention. This and other related advantages will be apparent to skilled persons from the description below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a size statistic report from Malvern Dynamic Light Scattering technology showing the dynamic light scattering pattern of collagen nanoparticles and that the formulation comprising the nanoparticles is monodispersed with an average diameter of 131nm with at least about 19% of the nanoparticles smaller than 100nm.

Figure 2 is a Transmission Electron Microscopy (TEM) Micrograph showing the morphology and size of collagen nanoparticles which is less than 100nm in diameter. Figure 3A is a graph of the dynamic light scattering pattern of an example of a collagen formulation comprising collagen liposomes (LP-23) showing the size distribution by intensity with an average diameter of 114.9nm.

Figure 3B is graph showing the zeta potential of an example of a collagen formulation comprising collagen liposomes (LP-23).

Figure 3C is a size statistic report from Malvern Dynamic Light Scattering technology showing the dynamic light scattering pattern of collagen liposomes and that the formulation comprising the liposomes is monodispersed with an average diameter of 114.9nm.

Figure 4A is a graph of the dynamic light scattering pattern of an example of a collagen formulation comprising collagen niosomes (NS-63) showing the size distribution by intensity with an average diameter of 331.Onm. Figure 4B is graph showing the zeta potential of an example of a collagen formulation comprising collagen niosomes (NS-63).

Figure 4C is a size statistic report from Malvern Dynamic Light Scattering technology showing the dynamic light scattering pattern of collagen niosomes and that the formulation comprising the niosomes is monodispersed with an average diameter of 331.0nm. Figure 5 is an image of gel electrophoresis showing the collagen purity of collagen used in preparation of formulations comprising collagen nanostructures with a diameter less than 100nm Figure 6 are images of confocal laser scanning microscopy of skin after application of formulations comprising collagen nanoparticles loaded with Rodamine showing penetration capacity of the collagen nanoparticles.

Figure 7 are enlarged images of confocal laser scanning microscopy of skin after application of formulations comprising collagen nanoparticles loaded with Rodamine showing penetration capacity of the collagen nanoparticles.

Figure 8 are images of Z sectioning of skin after application of formulations comprising collagen nanoparticles loaded with Rodamine showing penetration capacity of the collagen nanoparticles.

Figure 9A and B are gamma sciuntigraphic images of human volunteers after application of formulations comprising collagen liposomes on the back, pre-wash and post-wash respectively to show retention, uptake and penetration capacity of the collagen liposomes and niosomes.

Figure 9C and D are gamma sciuntigraphic images of human volunteers after application of formulations comprising collagen liposomes on the hands, pre-wash and post-wash respectively to show retention, uptake and penetration capacity of the collagen liposomes and niosomes.

Figure 10 shows the different means by which the collagen nanostructures according to any aspect of the present invention may permeate and enter the skin surface. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference. Definitions

For convenience, certain terms employed in the specification, examples and appended claims are collected here.

Unless defined otherwise, all technical and scientific terms used herein include the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. It is believed that one skilled in the art can, based upon the description herein, utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of ± 5%, ± 10%, ± 20%, ± 25% or ± 30% of the value specified. For example, "about 50nm" can in some embodiments carry a variation from 45nm to 55nm. In another embodiment, for example, a nanoparticle of about 100nm in diameter refers to a nanoparticle of 70-130nm in diameter. For any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof as well as the individual values making up the range, particularly integer values. A recited range (e.g., size of nanostructures) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more" and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

As will be understood by a skilled person, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and understood as being modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the present teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As used herein, the term "cold temperature" refers to a temperature below human body temperature. For example, cold temperature can be any temperature below 38°C. In particular, the temperature may be 30°C, 25°C, 20°C, 15°C, 10°C, 8°C, 5°C and the like.

By "comprising" is meant including, but not limited to, whatever follows the word comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or option of the listed elements.

As generally used herein, "drug" refers to any biologically active agent, including but not limited to classical small-molecule drugs, therapeutically effective proteins, lipids, polysaccharides, proteoglycans, and polynucleotides. The drug may be a therapeutic, a prophylactic, or a diagnostic agent.

As used herein, the term "enhanced penetration and/or retention" refers to improved permeation into the skin surface by the collagen nanostructure and retaining in the skin for longer period of time according to any aspect of the present invention and/or improved preservation of the nanostructure and its effects in a target region compared to other collagen nanostructures known in the art. In a non-limiting example, the collagen nanostructure according to any aspect of the present invention has been proven to penetrate the skin surface of humans and/or animals efficiently and effectively and/or is capable of maintaining its form and effect in the skin epithelium beneath the skin surface longer than the collagen nanostructures known in the art. The enhanced penetration and/or retention of the collagen nanostructures can be measured using confocal microscopy and/or in vitro permeation study taught in Examples 9 and 10 below. In particular, a collagen nanostructure may be said to have enhanced penetration and/or retention using in vitro permeation study if it has a maximum permeation of collagen of 1 % to 15%, 2% to 10%, 3% to 6% or the like and retention within the skin of 2% to 10%, 2% to 8%, 2% to 5% or the like. Other examples known in the art may also be used to measure penetration, retention of the nanostructures and/or human gammascintigraphic study.

As used herein, the term "formulation" refers to any nanostructure according to any aspect of the present invention intended for the administration of a_. pharmaceutical compound, or combination, including, but not limited to, any chemical or peptide, natural or synthetic, that is administered to a patient for medicinal purposes. Specifically, a formulation may comprise either a single nanostructure or a plurality of nanostructures.

As used herein, the term "liposome" refers to a particle comprising one or more lipid bilayers enclosing an interior, typically an aqueous interior. Thus, a liposome is often a vesicle formed by a bilayer lipid membrane. The liposomes according to any aspect of the present invention may by small, multilamellar or unilamellar. For example, the liposomes of the present invention are collagen liposome comprising at least one dimension having a size less than 300nm. In particular, the diameter of the liposome may be less than or equal to 300nm, less than 250nm, less than 200nm, less than 150nm, less than 100nm or less than 50nm.

As used herein, the term "micelle" refers to a collagen based aggregate of surfactant molecules dispersed in a liquid colloid. The micelle according to the present invention when in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic single tail regions in the micelle centre. This phase is caused by the insufficient packing issues of single tailed lipids in a bilayer.

As used herein, the term "monodisperse" refers to a collection of objects having the same size and shape. For example, a monodisperse of the nanoparticles according to any aspect of the present invention comprises nanoparticles of the same size and shape.

As used herein, the term "nanostructure" refers to an extremely small particle. The term "nanostructure" as used herein refers to an extremely small particle. The nanostructure may comprise at least one dimension having size < 1000 nm. For example, < 700nm, 500nm, 300nm, 250nm, 200nm, 150nm or 100nm, in particular, < 50nm and even more in particular, less than 50 nm. More in particular, the nanostructure may comprise at least one dimension of size < 25 nm, and even more in particular the nanostructure may comprise at least one dimension of size < 10 nm or < 5 nm. The dimension may refer to the average diameter of the nanostructure. The term "nanostructure" may be used to describe a nanoparticle, liposome, micelle or niosome etc. For example, the nanoparticle according to any aspect of the present invention may comprise at least one dimension having a size in the range of 1 to 200 nm. For example, 180nm, 150nm, 130nm or 100nm, in particular, 50nm and even more in particular, less than 50 nm. The nanoparticle may comprise at least one dimension of size 25 nm, and even more in particular the nanoparticle may comprise at least one dimension of size 10 nm or 5 nm. The dimension may refer to the average diameter of the nanoparticle.

As used herein, the term "niosome" refers to a non-ionic surfactant-based liposome. Niosomes are formed mostly by cholesterol incorporation as an excipient. Other excipients can also be used. In particular, the noisome according to any aspect of the present invention may comprise at least one dimension having a size less than 300nm. In particular, the diameter of the niosome may be less than or equal to 300nm, less than 250nm, less than 200nm, less than 150nm or less than 100nm.

As used herein, the term "water for injection" refers to water that is purified by distillation or two-stage reverse osmosis.

As used herein, the term "zeta potential" is a measure of the magnitude of the repulsion or attraction between particles. Its measurement brings detailed insight into the dispersion mechanism and is the key to electrostatic dispersion control thus can be used to determine stability. For example, Malvern Instruments' Zetasizer systems can be used to measure zeta potential measures.

A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books.

In the broadest aspect the present invention is directed towards the production of nanostructures of collagen. As such, any solubilised collagen known in the prior art can be used. These collagens can be extracted and purified from the connective tissue of various organs such as skin, bone, cartilage, tendon, and viscous of animals such as cows, pigs, birds, kangaroos, sheep and so forth by acidic solubilisation, alkaline solubilisation, neutral solubilisation and or enzymatic solubilisation, but also includes chemically-modified collagen and the like. Once isolated, the collagen can be processed to produce collagen nanoparticles.

In particular, the collagen nanostructure may have at least one dimension that is less than or equal to 300nm. The dimension may a diameter of a regularly shaped nanostructure (for example a sphere) or the largest dimension of an irregularly shaped nanostructure. More in particular, the dimension may be from 50nm to 200nm, from 50nm to 100nm, from 80nm to 250nm, from 80nm to 280nm, from 70nm to 290nm and the like.

The collagen nanostructure may be a nanoparticle, liposome, micelle, noisome and the like. In one example, the nanostructure may be a nanoparticle with a diameter less than or equal to 100nm, 80nm, 50nm or the like. In another example, the collagen nanostructure may be a liposome with a diameter less than 300nm, 200nm, 150nm, 100nm or the like. In a further example, the collagen nanostructure may be a niosome with a diameter less than less than 300nm, 200nm, 150nm, 100nm or the like. In particular, the collagen nanostructures according to any aspect of the present invention may be capable of enhanced penetration and/or retention across at least one biological surface relative to a control. The penetration and/or retention are a measure of the percentage of collagen nanostructures that pass across the biological surface and/or remain within a region beneath the biological surface. For example, the biological surface may be a skin surface of a human being and/or an animal and the region beneath the biological surface may be an epidermal region. In particular, the control may be a collagen nanostructure not according to any aspect of the present invention. More in particular, the control may be a collagen nanostructure with a diameter greater than 300nm.

The enhanced penetration and/or retention may be due to:

A. Nano size lipophilic vesicle settle down to close contact with the skin and carrying collagen might penetrate into the skin (trans-cellular pathway)

B. Permeation of collagen carrying nano-sized lipophilic liposome and niosome vesicles through stratum corneum without vesicle fusion and subsequent drug release (trans-cellular pathway)

C. Intact vesicle penetration through the intact skin that may also follow the pore transport system (Para-cellular pathway).

D. Hydration of the stratum corneum due to external water phase of the formulation results in high diffusivity of drug as droplet size approaches to molecular dispersion.

E. And most importantly, bigger hydrophilic collagen molecule encapsulated in the lipophilic vesicle that modulate the collagen structure in entrapment of the collagen with the aqueous phase during the formation of spherical vesicle. Furthermore, lipophilic vesicular encapsulated collagen due to the surface coating leads to the lipophilic nature that facilitates the permeation through stratum corneum.

F. In addition, follicular transport may also involve along with the other mechanism in collagen dermal deposition as shown in Figure 10.

According to another aspect, the present invention provides a collagen nanostructure formulation comprising a plurality of collagen nanostructures according to any aspect of the present invention where at least 70% of the nanostructures have a diameter less than 250nm. In particular, at least 70% of the nanostructures have a diameter less than 100nm, 80nm, 50nm or the like. More in particular, at least 75%, 80%, 85%, 90% or 95% of the nanostructures have a diameter less than 100nm, 80nm, 50nm or the like.

In one non-limiting example, the nanostructures may be nanoparticles, wherein at least 70% of the nanoparticles may have a diameter less than 300nm, 200nm, 100nm, 80nm or 50nm. In another example, the nanostructures may be liposomes, wherein at least 70% of the liposomes may have a diameter less than 300nm, 250nm, 200nm or 100nm. In another example, the nanostructures may be niosomes, wherein at least 70% of the niosomes may have a diameter less than 300nm, 250nm, 200nm or l OOnm.According to another aspect, the present invention provides a method of producing a collagen nanostructure and/or collagen nanostructure formulation according to any aspect of the present invention comprising the steps of:

(i) reducing at least one metal ion with at least one reducing agent;

(ii) adding at least one polymer to (i);

(iii) adding at least one collagen solution to (ii);

(iv) adding at least one cross-linker to (iii).

According to another aspect, the present invention provides a collagen nanostructure and/or formulation prepared according to the method of any aspect of the present invention.

According to a particular non limiting aspect, the process of the invention may involve preparing two independent solutions before mixing them, then adding a cross-linker solution to the mixture of solutions and then stirring the mixture, for example overnight, in order to achieve the collagen nanostructures of the present invention. In particular, the first solution may comprise a plurality of collagen particles. The plurality of collagen particles may be dissolved at cold temperature. On the other hand, the second solution may comprise a plurality of metallic nanostructures which may be prepared by reducing metallic ions with a reducing agent and in particular, capped by a polymer to prevent further growth.

Therefore, in a particular aspect, the process for producing a plurality of collagen nanostructures according to any aspect of the present invention may comprise the steps of:

(i) dissolving collagen (for example at cold temperature);

(ii) reducing metallic ions with a reducing agent;

(iii) adding a polymer to (ii);

(iv) mixing (i) and (iii);

(v) adding a cross-linker solution to (iv); and

(vi) stirring (v) (for example overnight).

The collagen used in the present invention includes but is not limited to low molecular weight collagen and/or hydrolyzed collagen. The collagen may be thoroughly dissolved by mixing in water for injection, particularly at the cold temperature in the range of 0-10°C. In particular, the collagen may be thoroughly dissolved by mixing at 5°C.

The collagen may then be mixed with metallic nanostructures. The metallic nanostructures include but are not limited to silver nanoparticles, gold nanostructures, iron nanostructures, copper nanostructures, zinc nanostructures, nickel nanostructures, lead nanostructures and the like. The metallic ions may be first reduced with a reducing agent before mixing with the collagen. The reducing agent includes but is not limited to Sodium borohydride, Hydrazine Hydrate and Lithium Aluminium Hydride. The metallic nanoparticles are capped by polymer to prevent further growth. The polymer includes but not limited to polyacrylic acid, poly vinyl alcohol, poly (N-isopropylacrylamide), poly (ethylenimine) poly (N-vinyl-2-pyrrolidone) and the like.

For example, the silver nanostructures may be prepared in an aqueous solution by reducing Ag⁺ ions with Sodium Borohydride. The silver nanostructures may then be capped by polyacrylic acid to prevent further growth.

The collagen may be mixed with the metallic nanoparticle by vortexing. A cross-linker may then be added to the mixture and stirred overnight. The cross-linker may include but is not limited to Malondialdehyde, EDCI {1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride}, Glutaraldehyde pentasodium tripolyphosphate (TPP) and the like.

In particular, the resultant collagen nanostructures may be in the form of liquid dry powder and/or granule. Dried collagen nanostructures can be obtained by a further step of drying. The drying process may include but is not limited to freeze-drying, desiccation by evaporation at low temperature spray-drying and the like.

A combination of various analytical techniques can be used to elucidate the size and structure of collagen nanostructures of the present invention. These techniques include UV-vis, Dynamic Light Scattering (DLS), Brunauer-Emmett-Teller (BET) specific surface area, Transmission Electron Microscopy (TEM), X-ray diffraction and the like. For example, TEM imaging may be used to verify the particle size and morphology characteristics, in conjunction with X-ray diffraction data for surfactant molecules thus being able to give greater insights to the supramolecular structure of the protective layer. SDP precipitation technique may be unique in being able to control the size of the produced nanostructures and TEM results may support the DLS sizing data and may demonstrate low aspect ratio particle morphologies.

According to a particular aspect, the process for producing collagen nanostructures according to any aspect of the present invention comprises the steps of:

(i) dissolving low molecular weight collagen (for example at cold temperature);

(ii) reducing Ag⁺ ions with sodium borohydride;

(iii) adding polyacrylic acid to (ii);

(iv) mixing (i) and (iii);

(v) adding Malondialdehyde to (iv); and

(vi) stirring (v) (for example overnight).

In an embodiment, the process for producing collagen nanostructures according to any aspect of the present invention comprises the steps of:

(i) dissolving low molecular weight collagen (for example at cold temperature);

(ii) reducing Ag⁺ ions with sodium borohydride;

(iii) adding polyacrylic acid to (ii);

(iv) mixing (i) and (iii);

(v) adding EDCI {1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride} to (iv); and

(vi) stirring (v) (for example overnight).

In another embodiment, the process for producing collagen nanostructures according to any aspect of the present invention comprises the steps of:

(i) dissolving low molecular weight collagen (for example at cold temperature);

(ii) reducing Ag⁺ ions with sodium borohydride;

(iii) adding polyacrylic acid to (ii);

(iv) mixing (i) and (iii);

(v) adding Glutaraldehyde to (iv); and

(vi) stirring (v) (for example overnight).

In a further embodiment, the process for producing collagen nanostructures according to any aspect of the present invention comprises the steps of:

(i) dissolving low molecular weight collagen (for example at cold temperature);

(ii) reducing Ag⁺ ions with sodium borohydride;

(iii) adding polyacrylic acid to (ii);

(iv) mixing (i) and (iii); (v) adding pentasodium tripolyphosphate to (iv); and

(vi) stirring (v) (for example overnight).

The collagen nanostructures of the invention may be suitable for preparation of compositions for topical or parenteral use. For parenteral use, said nanostructures may be administered in doses corresponding to an amount of collagen ranging from 0.01 to 5 mg/kg of body weight, more preferably ranging from 2 to 3 mg/kg.

In the compositions for topical ocular administration for example, the concentration of collagen nanostructures may range from 1 to 25% weight/volume. The nanostructures of the present invention may optionally contain an amount of viscosizing substance which may range from 0.1 to 0.4% by weight.

According to a further aspect, the present invention relates to the use of collagen nanostructures for incorporation and delivery of therapeutic agents or drugs. Any drug that may be capable of being transformed into a microstructure or nanostructure material together with collagen may be formed into nanostructures using the method according to any aspect of the present invention. The drug may be in either small molecule or macromolecular forms. The drug may be of low solubility in bodily fluids or completely soluble in bodily fluids. For example, the extremely small particle size of the collagen nanostructures in the present invention can be useful in delivery of a suitable drug as an aerosol to the nasal passages and sinuses, or to the lungs and the like. The method may be also useful in preparation of dosage forms of shear-sensitive drugs, such as proteins and nucleic acids. A large number of drugs which are known and may be used in conjunction with the nanostructures of the present invention, are listed in standard compendia such as the Merck Index and the Physicians' Desk Reference.

The collagen nanostructures may be used alone, or may be coated with one or more surface-active agents ("surfactants"), polymers, adhesion promoters, or other additives or excipients. The nanaoparticles of the present invention may be incorporated into tablets or capsules or other dosage forms, or encapsulated. Many different excipients may be commonly used in drug formulations. Classes of excipients include, but are not limited to, "tableting aids, disintegrants, glidants, antioxidants and other preservatives, enteric coatings, taste masking agents, and the like. References describing such materials are readily available to and well-known by the practitioners in the art of drug formulations. The excipients may be added during any of the steps described below for including surfactants in the nanostructures. For example, the excipient may be added during the formation of the nanostructure; during the dispensing of the nanostructures to form a dosage form; or during the administration of the nanostructures. The selection of the additives or excipients may be determined in part by the projected route of administration. Any of the conventional routes (e.g. inhalation, oral, rectal, vaginal, topical, parenteral and the like) may be suitable for, and may be enhanced by, the use of the nanostructure drug formulations. Suitable formulations include but are not limited to oral formulations, aerosols, topical formulations, parenteral formulations, and implantable compositions. In particular, the nanostructure drug formulations may be particularly suitable for delivering hydrophobic and other poorly-soluble drugs, such as those in bioavailability classes II and IV, by oral or aerosol administration, thereby replacing a parenteral route of administration currently being used. Optionally, the collagen nanostructures may contain a surfactant to eliminate or reduce aggregation of the particles. The surfactant may adhere to the surface of the nanoparticles. Typically, a surfactant may facilitate the dispersion of the nanostructures in any or all of the initial non-solvent mixtures in which the particle may be formed, the medium in which the nanostructures may be taken up for administration, and the medium (e.g. gastrointestinal fluid) into which the particle may be later delivered.

Any surfactant may be useful for use with the collagen nanostructures. Suitable surfactants include small molecule surfactants, often called detergents, macromolecules (i.e. polymers) and the like. The surfactant may also contain a mixture of surfactants. In formulations for parenteral administration, the surfactant may be preferably one that is approved by the FDA for pharmaceutical uses. In formulations for non-parenteral administration, the surfactant may be one that is approved by the FDA for use in foods or cosmetics. The surfactant may be present in any suitable amount. For example, effective surfactants may be present as only a small weight fraction of the collagen nanostructures, such as from 0.1 % to 10% (wt of surfactant/weight of the collagen). Alternatively, larger proportions of surfactant may be needed, thus the surfactant may be present in a weight percent of 20%, 50% or up to about 100% of the weight of the collagen, particularly when the particles are small and the total surface area may be accordingly large. Suitable polymers that may be used in the present invention as a surfactant may include soluble and water-insoluble, and biodegradable and non-biodegradable polymers, including but not limited to hydrogels, thermoplastics, and homopolymers, copolymers and blends of natural and synthetic polymers. Representative polymers include hydrophilic polymers, such as those containing carboxylic groups, including polyacrylic acid. Bioerodible polymers may include polyanhydrides, poly(hydroxyl acids) and polyesters, as well as blends and copolymers thereof. Representative bioerodible poly(hydroxyl acids) and copolymers thereof include but are not limited to poly(lactic acid), poly(glycolic acid), poly(hydroxybutyric acid), poly (hydroxyvaleric acid), poly (caprolactone) , poly (lactide-co-caprolacto- ne), poly(lactide-co-glycolide) and the like. Polymers containing labile bonds, such as polyanhydrides and polyorthoesters, can also be used optionally in a modified form with reduced hydrolytic reactivity. Positively charged hydrogels, such as chitosan, and thermoplastic polymers, such as polystyrene can also be used.

Representative natural polymers which also can be used in any aspect of the present invention include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, and polysaccharides such as dextrans, polyhyaluronic acid and alginic acid. Representative synthetic polymers include polyphosphazenes, polyamides, polycarbonates, polyacrylamides, polysiloxanes, polyurethanes and copolymers thereof. Celluloses also can be used. As defined herein the term "celluloses" includes naturally occurring and synthetic celluloses, such as alkyl celluloses, cellulose ethers, cellulose esters, hydroxyalkyl celluloses and nitrocelluloses. Exemplary celluloses include ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose triacetate and cellulose sulfate sodium salt.

Polymers of acrylic and methacrylic acids or asters and copolymers thereof may be used in any aspect of the present invention. Representative polymers which can be used include but are not limited to poly (methyl methacrylate) , poly(ethyl methacrylate), poly (butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and the like.

Other polymers which can be used include but are not limited to polyalkylenes such as polyethylene and polypropylene; polyarylalkylenes such as polystyrene; poly(alkylene glycols), such as poly(ethylene glycol); poly(alkylene oxides), such as poly(ethylene oxide); and poly(alkylene terephthalates), such as poly(ethylene terephthalate) and the like. Polyvinyl polymers can also be used, which, as defined herein includes polyvinyl alcohols, polyvinyl ethers, polyvinyl esters and polyvinyl halides. Exemplary polyvinyl polymers include poly (vinyl acetate), polyvinyl phenol and polyvinylpyrrolidone and the like.

Polymers which alter viscosity as a function of temperature, shear or other physical forces may also be used. Poly(oxyalkylene) polymers and copolymers such as poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) or poly(ethylene oxide)-poly(butylene oxide) (PEO-PBO) copolymers, and copolymers and blends of these polymers with polymers such as poly(alpha-hydroxy acids), including but not limited to lactic, glycolic and hydroxybutyric acids, polycaprolactones, and polyvalerolactones, can be synthesized or commercially obtained. For example, polyoxyalkylene copolymers are described in U.S. Pat. Nos. 3,829,506; 3,535,307; 3,036,118; 2,979,578; 2,677,700; and 2,675,619.

These polymers can be obtained from sources such as Sigma Chemical Co., St. Louis, Mo.; Polysciences, Warrenton, Pa.; Aldrich, Milwaukee, Wis.; Fluka, Ronkonkoma, N.Y.; and BioRad, Richmond, Calif., or can be synthesized from monomers obtained from these or other suppliers using standard techniques.

Representative examples of wetting agents include but are not limited to mannitol, dextrose, maltose, lactose, sucrose, casein, lecithin (phosphatides), gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers (e.g., macrogol ethers such as cetomacrogol 1000), polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters (e.g., TWEEN), polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcel!ulose, hydroxyethylcellulose, hydroxy propylcellulose, hydroxypropylmethylcellulose phthlate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone (PVP) and the like.

Preferred dispersants include hydrophilic polymers and wetting agents. The amount of dispersant in the formulation may be less than about 80%, more preferably less than about 75%, by weight of the formulation In particular, the polymer for use as a dispersant may be polyvinylpyrroidone. One or more surfactants or other excipients can be added to the collagen and/or collagen-drug combination in a number of ways. A surfactant may be applied at one or more of several steps in the process of producing and dispensing nanostructures of the present invention. For example, the surfactant may be present in the initial solution of collagen or other nanostructure-forming material. In another example, the surfactant may be present in the solvent that is mixed with the collagen or collagen-drug combination to form the nanostructures. The surfactant may be added to a drug solution before precipitation with the collagen. In particular, this method is particularly useful for small- molecule surfactants. Any medical or veterinary condition that can be treated with collagen or a drug carried by the collagen nanostructures of the present invention and may be treated using the nanostructure collagen. In particular, the formulation may be administered to treat a disease such as cancer and the like or to administer an oral vaccine, or for any other medical or nutritional purpose requiring uptake through the mucosa of the drug or bioactive to be delivered.

Collagen nanostructures may be administered to a patient by a variety of routes. For example, these include, without limitation, oral delivery to the tissues of the oral cavity, the gastrointestinal tract and by absorption to the rest of the body; delivery to the nasal mucosa and to the lungs (pulmonary); delivery to the, skin, or transdermal delivery; delivery to other mucosa and epithelia of the body, including the reproductive and urinary tracts (vaginal, rectal, urethra); parenteral delivery via the circulation; delivery from locally implanted depots or devices and the like. Aspects of the invention will now be described in greater detail by reference to the following non-limiting examples. Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. EXAMPLES

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. Example 1

50ml of water for injection was poured in a round bottom flask and 0.001 gms of low molecular weight collagen (Ovicoll from Holista colltech Ltd.) was added and completely dissolved by mixing at temperature 0-5° C. This solution was termed as Solution TV.

In another beaker, silver nanoparticles were prepared in aqueous solution by reducing Ag⁺ ions with sodium borohydride. The metallic silver produced is immediately capped by polyacrylic acid to prevent further growth.

The silver nanoparticles were prepared in aqueous phase by chemical reduction of silver salt solution using sodium borohydride in the presence of polyacrylic acid as a capping agent. 800μΙ of silver nitrate solution (5% w/v) and 800μΙ of polyacrylic acid (50%w/v) were thoroughly mixed in 40 ml double distilled water at 4° C with constant stirring. After mixing the solutions, the mixture was further stirred for another hour. 100 ml of 0.01 M ice- cooled NaBH₄ solution was then added to the stirred mixture and the resultant solution became a light green colour. The resultant solution was further stirred for about two hours at 4° C. After stirring was completed, the solution was dialyzed through 24 kD dialysis bag for 3-4 hours against distilled water and the solution changed from light green to yellow in colour. The dialyzed solution containing silver particles were diluted to get silver nanoparticles solution of known concentration to carry out the further reactions.

In the next step the 2ml of silver nanoparticles dispersion was added to solution 'A' and thoroughly mixed by vortexing. After mixing, 2mg of Malondialdehyde ( Sigma Aldrich, India), was added and stirred the solution overnight. The resultant solution was characterized by DLS and TEM.

Table 1 Data produced from DLS study of nanoparticles i.e. average particle size and polydispersity index of that population Diameter (nm) % Intensity Width (nm)

Peak l : 131.2 100.0 41.64

Peak 2: 0.000 0.0 0.000

Peak 3: 0.000 0.0 0.000

Table 2 Characterization of Nanocollagen by DLS

The characterization of the resultant solution by DLS is shown in Tables 1 and 2 and which indicate that the size/diameter of nanoparticles was about 131.2 with 100% intensity meaning the sample was highly monodisperse. This is illustrated by the graph in Figure 1.

Figure 2 shows that the morphology and size of nanocollagen was less than 100nm in diameter and well distributed.

Example 2

In a typical set of reaction, 50ml of water for injection was taken in a round bottom flask and O.OOIgms of low molecular weight collagen was added and thoroughly dissolved by mixing at temperature 0-5°C. This solution is termed as Solution 'Α'.

In a separated beaker, silver nanoparticles were prepared in aqueous solution by reducing Ag+ ions with sodium borohydride. The metallic silver produced is immediately capped by polyacrylic acid to prevent further growth.

The detailed synthetic procedure of silver nanoparticles is as follows: The particles were prepared in aqueous phase by chemical reduction of silver salt solution using sodium borohydride in the presence of polyarylic acid as capping agent. 800μΙ of silver nitrate solution (5% w/v) and 800μΙ of polyacrylic acid (50%w/v) were thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring. After mixing the solutions the mixture was further stirred for another hour. 100 ml of 0.01 M ice-cooled NaBH₄ solution was then added to the stirred mixture when it became light green in colour. The resultant solution was further stirred for two hours at 4°C. After stirring was completed, the solution was dialyzed through 24 kD dialysis bag for 3-4 hours against distilled water when the solution was changed from light green to yellow. The dialyzed solution containing silver particles were diluted to get silver nanoparticles solution of known concentration to carry out the further reactions.

In the next step the 2ml of silver nanoparticles dispersion was added to solution 'A' and thoroughly mixed by vortexing. After mixing 50μΙ of Glutaraldehyde was added and stirred the solution for overnight. The resultant solution was characterized by DLS and TEM. Similar results as those obtained in Example 1 were obtained. Example 3

In a typical set of reaction, 50ml of water for injection was taken in a round bottom flask and 0.001 gms of low molecular weight collagen was added and thoroughly dissolved by mixing at temperature 0-5°C. This solution is termed as Solution TV.

In a separated beaker, silver nanoparticles were prepared in aqueous solution by reducing Ag+ ions with sodium borohydride. The metallic silver produced is immediately capped by polyacrylic acid to prevent further growth.

The detailed synthetic procedure of silver nanoparticles is as follows: The particles were prepared in aqueous phase by chemical reduction of silver salt solution using sodium borohydride in the presence of polyarylic acid as capping agent. δΟΟμΙ of silver nitrate solution (5% w/v) and 800μΙ of polyacrylic acid (50%w/v) were thoroughly mixed up in 40 ml double distilled water at 4°C with constant stirring. After mixing the solutions the mixture was further stirred for another hour. 100 ml of 0.01 M ice-cooled NaBH4 solution was then added to the stirred mixture when it became light green in colour. The resultant solution was further stirred for two hours at 4°C. After stirring was completed, the solution was dialyzed through 24 kD dialysis bag for 3-4 hours against distilled water when the solution was changed from light green to yellow. The dialyzed solution containing silver particles were diluted to get silver nanoparticles solution of known concentration to carry out the further reactions.

In the next step the 2ml of silver nanoparticles dispersion was added to solution 'A' and thoroughly mixed by vortexing. After mixing 3mg of EDCI {1 -(3-dimethylaminopropyl)3- ethylcarbodiimide hydrochloride} was added and stirred the solution for overnight. The resultant solution was characterized by DLS and TEM. Similar results as those obtained in Example 1 were obtained showing that different cross-linkers may be used to obtain the nanostructures of the present invention.

Example 4

0.50 ml of Tween80/polysorbate80/queen80 was added in the 20.0 ml of 0.015% solution of collagen in water with constant stirring for about one hour. In order to crosslink the nanoparticles, 0.10 ml of Malenodialdehyde (or any cross linker which forms Schiffs base with NH₂ group of collagen) was added to the solution and the stirring continued for another 30 minutes. Finally 1.0 ml of 10% sodium metabisulphite was added, stirred for another 30 minutes and was kept for twelve hours. The entire solution was, then, dialyzed against water with two changes at 4°C for 24hrs followed by another 24hrs against normal saline with two changes at 4°C. The dialyzed solution was preserved at 4°C and directly used for further characterization using DSL. The results are shown in Table 2 below and Figure 3.

Table 3 Characterization of Nanocollagen by DLS Example 5

Liposomal formulations were prepared using ethanol injection method as mentioned by Pons et al, 1993 with slight modification. Appropriate ratio of phospholipid and cholesterol was dissolved in absolute ethanol (total concentration of mixture was varied from 10mg/mL to 100mg/mL); 1 mL bolus and rapid injection of organic phase was then injected in the aqueous phase (collagen solution) through 30 gauge syringe followed by stirring for 1 h to remove the ethanol completely. Finally, all the formulations were sonicated for desired time [amplitude 35% and pulse 3sec: 5sec (on: off)] maintaining the temperature below 350C.Vesicle formation becomes evident on the appearance of the characteristic opalescence of colloidal dispersions. Two different examples (A) and (B) are provided below.

(A) Accurately weighed Phospholipon 90H (400mg) and Cholesterol (10mg) were mixed in 10mL of ethanol (99%) in 15mL falcon tube, vortexed for 5min to make clear organic phase. In a 50mL beaker 20mL of aqueous collagen solution (concentration 0.5mg/mL) is taken and kept on stirring at 500rpm while maintaining temperature of 30±0.50C on a magnetic stirrer. 1 mL bolus and rapid injection of organic phase was then injected in the aqueous phase (collagen solution) through 30gauge syringe followed by continuous stirring for I n to remove the ethanol completely. Finally, the mixture was sonicated for 1min [amplitude 35% and pulse 3sec: 5sec (on: off)] maintaining the temperature below 350C. Liposomes prepared were found to be 293±2.41 nm in size (PDI=0.051) using DLS. (B) Accurately weighed Phospholipon 90H (240mg) and Cholesterol (60mg) were mixed in 10mL of ethanol (99%) in 15mL falcon tube, vortexed for 5min to make clear organic phase. In a 50mL beaker 20mL of aqueous collagen solution (concentration 0.5mg/mL) is taken and kept on stirring at SOOrpm while maintaining temperature of 30±0.50C on a magnetic stirrer. 1 mL bolus and rapid injection of organic phase was then injected in the aqueous phase (collagen solution) through 30gauge syringe followed by continuous stirring for 1 h to remove the ethanol completely. Finally, the mixture was sonicated for 3min [amplitude 35% and pulse 3sec: 5sec (on: off)] maintaining the temperature below 350C. The developed formulation was in the size value of 1 15±1.19nm (PD 0.073) using DLS as shown in Figure 3.

Example 6

Niosomes were prepared by using ethanol injection method with slight modification (Shaikh et al, 2010). In this method Span60 or Span20, tween 20, 40, 60, 80, Cremophore EL.RH, Triton X 100, Labrasol, Lauroglycol 90, Labrafil M and combinations and Cholesterol (Sigma Aldrich, SD fine chemicals, Delhi,, Colorcon, India) in the different ratios were dissolved in ethanol (total concentration of mixture was varied from 10mg/ml_ to 150g/ml_) to form organic phase. An aqueous phase (concentration 0.5mg/mL to 70mg/mL of collagen) in 20mL of phosphate buffer (pH 7). The aqueous phase was kept on a magnetic stirrer at temperature of 35±0.5°C and 500rpm. 1 ml_ bolus and rapid injection of organic phase was then injected in the aqueous phase through 30gauge syringe followed by stirring for 1 h to remove the ethanol completely. To maintain the uniformity of size in dispersion, the developed niosomes were sonicated by probe sonicator [Vibra-Cell™ VC 750; Sonics, USA] for 0.5min-30min at amplitude 35% and pulse 3sec: 5sec (on: off) maintaining the temperature below 35°C. Two different examples (A) and (B) are provided below.

(A) Accurately weighed Span 60 (200mg) and Cholesterol (200mg) were mixed in 10mL of ethanol (99%) in 15ml_ falcon tube, vortexed for 5min with intermittent heating in water bath to make clear organic phase. In a 50ml_ beaker 20ml_ of aqueous collagen solution (concentration 0.5mg/ml_) is taken and kept on stirring at 500rpm while maintaining temperature of 30±0.5°C on a magnetic stirrer. 1 mL bolus and rapid injection of organic phase was then injected in the aqueous phase (collagen solution) through 30gauge syringe followed by continuous stirring for 1h to remove the ethanol completely. Finally, the mixture was sonicated for 1 min [amplitude 35% and pulse 3sec: 5sec (on: off)] maintaining the temperature below 35°C. Niosomes prepared were measured using DLS and found to be of the size 50nm-300nm as shown in Figure 4.

(B) Accurately weighed Span 60 (150mg) and Cholesterol (100mg) were mixed in 10ml_ of ethanol (99%) in 15mL falcon tube, vortexed for 5min with intermittent heating in water bath to make clear organic phase. In a 50ml_ beaker 20ml_ of aqueous collagen solution (concentration 0.5mg/ml_) is taken and kept on stirring at 500rpm while maintaining temperature of 30±0.5°C on a magnetic stirrer. 1 mL bolus and rapid injection of organic phase was then injected in the aqueous phase (collagen solution) through 30gauge syringe followed by continuous stirring for 1 h to remove the ethanol completely. Finally, the mixture was sonicated for 3min [amplitude 35% and pulse 3sec: 5sec (on: off)] maintaining the temperature below 35°C. Niosomes prepared were measured using DLS and found to be of the size 75nm-125nm.

Example 7

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, was used on collagen to see the integrity of collagen after making a formulation. The collagen was found to be intact as shown in Figure 5.

Example 8

The results of the confocal microscopy done to study the penetration capacity of nanostructures into the skin is given in Figures 6-8. In particular, to evaluate the retention, uptake and the penetration capacity of Rodamine (Sigma Aldrich, India) loaded nanoparticles into the skin epithelium, skin specimens from ex vivo perfusion experiments were directly mounted, epithelial side up, on a glass slide and examined without further tissue processing by CLSM (Olympus FluoView FV 1000, Hamburg, Germany). Samples were excited with green helium neon 543 nm laser beam. Images were taken through a 20X oil objective, assembled in an integral image processor and displayed on a digital video monitor. To confirm the penetration of nanoparticles stacks of serial 4.4 pm optical sections were captured along the Z-axis. The presence of round fluorescent spots in- between as well as inside the cells as shown in Figures 6-8 suggested that the uptake of Nanoplexes via paracellular (movement between the cells through 'leaky' tight junctions) and transcellular (movement across plasma membrane of the cells) routes.

Example 9

In vitro permeation study was performed for both liposomal and niosomal formulations using Franz diffusion cell through hairless mice skin. First of all collagen was labeled using technetium 99 (^99mTc), which was then used for preparation of liposome and niosomes. 2ml_ of formulation was put in the donor chamber, 15mL of phosphate buffer (pH 5.8) was taken in the receptor chamber and the system was kept on magnetic stirrer at speed 100rpm and temperature 35±0.5°C. 1 ml_ of sample was taken out at different time interval and replaced by fresh buffer. Activity counts of all the samples were taken using gamma counter. Amount of collagen permeated was also determined using Ultra Performance Liquid Chromatography (UPLC). Concentration determined using both activity counting method as described in Mohit Gulati ef a/., 2005 and UPLC was correlated to confirm that activity permeated is the actual measure of drug permeated. The activity remained within the skin was also determined to calculate the retention of collagen in the skin. Control experiments were also performed using Tc-labelled collagen solution. Control was collagen solution labeled with ^99mTc. The results are provided in Table 4.

Table 4 Results of in vitro permeation study

As can be seen, the niosomes and liposomes made according to the method of the present invention have better permeation of collagen and retention within the skin. In both the cases of noisome and liposome smaller size formulation that is 100nm shows more permeation across the skin and less retention in the skin while formulation of 300 nm shows higher retention in the skin and less permeation across the skin.

Example 10

After taking approval from ethical committee Collagen nano formulations (liposomal as well niosomal formulations) were evaluated on healthy human subjects (male and female) for retention, uptake and the penetration capacity into the skin epithelium by using the gamma scintigraphy. The collagen was labeled using ^99mTc, which was then used for preparation of liposome and niosomes. The formulations were then applied on the hands at specific spots as per protocol. Pre-washing and post-washing images of the spots were taken. Percentage Skin deposition of the collagen were calculated for both liposomes as well as niosome. Negligible amount of collagen were found in the blood sample, when

0.5.l_ of blood was observed for activity count. The Liposomal and niosomal formulations respectively showed around 300% and 350% increased skin retention of collagen in comparison to direct application as shown in Figure 9.

REFERENCES

1. U.S.Pat. No 3,829,506;

2. U.S.Pat. No 3,535,307; 3. U.S.Pat. No 3,036,118;

4. U.S.Pat. No 2,979,578;

5. U.S.Pat. No 2,677,700;

6. U.S.Pat. No 2,675,619;

7. Mohit Gulati et al., International Journal of Nanomanufacturing, 20(3): 72-76, 2005;

8. Pons et al., International Journal of Pharmaceutics, 95 (1-3): 51-56, 1993;

9. Shaikh et al, Drug Dev Ind Pharm., 36(8):946-53, 2010.

Claims

1. A collagen nanostructure with a diameter less than 300nm.

2. The collagen nanostructure according to claim 1 , wherein the diameter is from 50nm to 200nm.

3. The collagen nanostructure according to either claim 1 or 2, wherein the diameter is from 50nm to 100nm.

4. The collagen nanostructure according to any one of the preceding claims, wherein the nanostructure is a nanoparticle, liposome, micelle or niosome.

5. The collagen nanostructure according to claim 1 , wherein the nanostructure is a nanoparticle with a diameter less than 100nm.

6. The collagen nanostructure according to claim 1 , wherein the nanostructure is a liposome with a diameter less than 100nm.

7. The collagen nanostructure according to claim 1 , wherein the nanostructure is a liposome with a diameter less than 300nm.

8. The collagen nanostructure according to claim 1 , wherein the nanostructure is a niosome with a diameter less than 100nm.

9. The collagen nanostructure according to claim 1 , wherein the nanostructure is a niosome with a diameter less than 300nm.

10. The collagen nanostructure according to any one of the preceding claims, wherein the nanostructure is capable of enhanced penetration and/or retention effect across at least one biological surface relative to a control.

1 1. The collagen nanostructure according to claim 10, wherein the control is a collagen nanostructure with a diameter greater than 300nm.

12. A collagen nanostructure formulation comprising a plurality of collagen nanostructures according to any one of claims 1 to 11 wherein at least 70% of the nanostructures have a diameter less than 250nm. .

13. The collagen nanostructure formulation according to claim 12, wherein the nanostructures are nanoparticles, wherein at least 70% of the nanoparticles have a diameter less than 100nm.

14. The collagen nanostructure formulation according to claim 12, wherein the nanostructures are liposomes, wherein at least 70% of the liposomes have a diameter less than 100nm.

15. The collagen nanostructure formulation according to claim 12, wherein the nanostructures are liposomes, wherein at least 70% of the liposomes have a diameter less than 250nm.

16. The collagen nanostructure formulation according to claim 12, wherein the nanostructures are niosomes, wherein at least 70% of the niosomes have a diameter of less than 100nm.

17. The collagen nanostructure formulation according to claim 12, wherein the nanostructures are niosomes, wherein at least 70% of the niosomes have a diameter of less than 300nm.

18. A method of producing a collagen nanostructure according to any one of claims 1 to 11 or a method of producing a collagen nanostructure formulation according to any one of claims 12 to 17 comprising the steps of:

(i) reducing at least one metal ion with at least one reducing agent;

(ii) adding at least one polymer to (i);

(iii) adding at least one collagen solution to (ii);

(iv) adding at least one cross-linker to (iii).

19. The method according to claim 18, wherein the metal ion is silver.

20. The method according to either claims 18 or 19, wherein the polymer is selected from the group consisting of polyacrylic acid, poly vinyl alcohol, poly (N- isopropylacrylamide), poly (ethylenimine)and poly (N-vinyl-2-pyrrolidone).

21. The method according to any one of claims 18 to 20, wherein the cross linker is selected from the group consisting of Malondialdehyde, EDCI {1-(3- dimethylaminopropyl)3-ethylcarbodiimide hydrochloride}, Glutaraldehyde and pentasodium tripolyphosphate.

22. The method according to any one of claims 18 to 21 , wherein the reducing agent is selected from the group consisting of Hydrazine Hydrate, Lithium Aluminium Hydride and Sodium Borohydride.

23. The method according to any one of claims 18 to 22, wherein the method comprises a further step of stirring overnight.

24. A collagen nanostructure prepared according to the method of any one of claims 18 to 23.

25. A collagen nanostructure formulation prepared according to any one of claims 18 to 23.

26. A method of enhancing penetration and retention effect of at least one active agent in a cell, the method comprising administering the nanostructure according to any one of claims 1 to 10 or formulation according to any one of claims 1 1 to 17 in combination with the active agent to the cell.

27. A method of treating a disease or disorder comprising the step of administering a collagen nanostructure according to any one of claims 1 to 11 or a collagen nanostructure formulation according to any one of claims 12 to 17 to a subject in need thereof.

28. The method according to claim 27, wherein the disease or disorder is a skin condition.

29. A collagen nanostructure according to any one of claims 1 to 10 or a collagen nanostructure formulation according to any one of claims 11 to 17 for use in medicine.

30. A collagen nanostructure according to any one of claims 1 to 10 or a collagen nanostructure formulation according to any one of claims 1 1 to 17 for use in treating a disease.

31. The collagen nanostructure and/or formulation according to claim 30, wherein the disease or disorder is a skin condition.

32. Use of a collagen nanostructure according to any one of claims 1 to 10 or a collagen nanostructure formulation according to any one of claims 1 1 to 17 in the preparation of a collagen nanoparticle formulation for the treatment of a disease.

33. The use according to claim 32, wherein the disease is a skin condition.