CELLULOSE-BASED TOPICAL FORMULATIONS

Abstract

The technology concerns topical formulations including nanocellulose materials for application on a skin or tissue of a subject.

Claims

1.-40. (canceled)

41. A topical formulation comprising at least one nanocellulose and optionally at least one cosmetically or pharmaceutically acceptable carrier, wherein the at least one nanocellulose is selected from nanofibrilar cellulose (NFC), oxidized NFC, crystalline nanocellulose (CNC) and oxidized CNC.

42. The formulation according to claim 41, provided as a film of the at least one nanocellulose on a skin region of a subject.

43. The formulation according to claim 41, the formulation comprising CNC or an oxidized form thereof.

44. The formulation according to claim 41, wherein the oxidized CNC is a product of oxidation of CNC with TEMPO.

45. The formulation according to claim 41, the formulation being in a form of a solution or a suspension comprising particles or flakes of the at least one nanocellulose.

46. The formulation according to claim 41, the formulation being in the form of a gel, an ointment, an emulsion, a thick cream, a liniment, a balsam, a lotion, a foam, a mask, a shampoo, tonic means, a cleaner, a spray, a hair spray, ear drops, eye drops, a conditioner, a roll-on, a powder including liquid powder, compact powder, cosmetic pencil, wet wipes or an application cloth.

47. The formulation according to claim 41, being contained in an applicator.

48. The formulation according to claim 47, wherein the applicator is a roller-ball applicator (a roll-on), a dispensing device for a cream or an ointment, a spray, a disposable or non-disposable applicator cloth, or a tissue or membrane.

49. A method for preventing or ceasing interaction of an allergen with a subject's skin region, the method comprising applying to the subject's skin region a formulation according to claim 41.

50. The method according to claim 49, wherein the formulation is applied by using a gel, an ointment, an emulsion, a thick cream, a liniment, a balsam, a lotion, a foam, a mask, a shampoo, tonic means, a cleaner, a spray, a hair spray, ear drops, eye drops, a conditioner, a roll-on, a powder including liquid powder, compact powder, cosmetic pencil, wet wipes or an application cloth comprising the formulation.

51. The method according to claim 49, wherein the formulation is applied by using an applicator comprising the formulation.

52. The method according to claim 49, for preventing or inhibiting a contact between an allergen and a skin region or binding of an allergen to an IgE epitope.

53. The method according to claim 49, wherein the formulation is applied onto a subject's skin region prior to exposure to an allergen or to a subject susceptible to having an allergic reaction.

54. The method according to claim 53, wherein the allergen is capable of inducing, promoting, or stimulating allergy.

55. The method according to claim 49, for preventing an allergen-mediated disease or disorder or form arresting deterioration of an existing condition due to continued exposure to the allergen.

56. The method according to claim 55, wherein the disease or disorder is caused by binding of IgE to an allergen causing immediate Type I allergic reaction.

57. The method according to claim 56, wherein the disease or disorder is selected amongst inflammatory, allergic and non-allergic diseases or disorders of the skin.

58. The method according to claim 57, wherein the disease or disorder is selected from hives (urticaria), eczema, angioedema, onchocercal dermatitis, dermatitis, atopic dermatitis, contact dermatitis and swelling.

59. A method for preventing atopic dermatitis in a subject, the method comprising applying to a skin region of the subject an amount of a formulation according to claim 41.

60. An applicator comprising a formulation according to claim 41.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0148] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0149] FIG. 1. SDS-PAGE analysis of Resilin-CBD/CNC binding assay at pH 8. Resilin-CBD/CNC at weight ratios of 10:1. Control: pure Resilin-CBD T: Total resilin-CBD/CNC prior CENTRICON separation R: retentate post CENTRICON separation P: peremeate post CENTRICON separation.

[0150] FIG. 2. SDS-PAGE analysis of Resilin-CBD/CNC binding assay at pH 5.5. Resilin-CBD/CNC at weight ratios of 10:1. T: Total resilin-CBD/CNC prior CENTRICON separation R: retentate post CENTRICON separation P: peremeate post CENTRICON separation.

[0151] FIG. 3. SDS-PAGE analysis of Ambrosia soluble pollen proteins /CNC binding assay at pH 7.5. in v:v ratio of 1:3 and 1:10 for Ambrosia soluble pollen proteins: OXCNC and 1:3 v:v ratio for Ambrosia soluble pollen proteins: CNC T: Total Ambrosia soluble pollen proteins/CNC prior CENTRICON separation. R: retentate post CENTRICON separation. P: peremeate post CENTRICON separation. Control: pure Ambrosia soluble pollen proteins.

[0152] FIGS. 4A-C. Binding assays showing Lolium perenne pollen binding to different forms of cellulose, including oxidized cellulose and microcrystalline cellulose (FIG. A), oxidized cellulose (FIG. B) and microfluidized CNC (FIG. C).

DETAILED DESCRIPTION OF EMBODIMENTS

Carbohydrate Binding Modules (CBMs)

[0153] One of the mechanisms that nature evolved is Carbohydrate Binding Modules (CBMs) which bind structural proteins to polysaccharide scaffolds such as cellulose in the plant kingdom and chitin in the animal kingdom respectively.

[0154] With respect to cellulose, during the course of evolution, many microorganisms developed an array of carbohydrate degrading enzymatic complexes termed “Cellulosomes”. Apart from the hydrolytic units, these complexes usually contain Carbohydrate Binding Modules (CBMs) that anchor the hydrolytic enzymatic complexes to the cellulose, many allergens, such as grass and dust mite, contain a common CBM motif.

Production of Crystalline Nano Cellulose

[0155] Currently, CNCs are mainly produced by acid hydrolysis/heat controlled techniques, with sulfuric acid being the most utilized acid. Extraction of the crystals from cellulose fibers involves selective hydrolysis of amorphous cellulose regions, resulting in highly crystalline particles with source dependent dimensions, e.g., 5-20 nm×100-500 nm for plant source CNCs. Sulfuric acid hydrolysis grafts negatively charged sulfate half-ester groups onto the surface of the particles, which act to prevent aggregation in aqueous suspensions due to electrostatic repulsion between particles. Furthermore, the rod-like shape of CNCs leads to concentration-dependent liquid crystalline self-assembly behavior.

TEMPO-Mediated Oxidation of CNCs

[0156] Total of 45 gr (4 mmol of glucosyl units) of cellulose nanocrystals were suspended in water (3.46 L) containing 692 mg of 2,2,6,6-tetramethyl-1-piperidinyloxyradical (TEMPO, 0.065 mmol) and 13.84 gr of sodium bromide (1.9 mmol) at room temperature for 30 min The TEMPO-mediated oxidation of the cellulose nanocrystals was initiated by slowly adding 734.37 mL of 6% NaClO (8.6 mmol) over 20 min at room temperature under gentle agitation. The reaction pH was monitored using a pH meter and maintained at 10 by incrementally adding 0.5 M NaOH. When no more decrease in pH was observed, the reaction was considered complete. About 346 mL of methanol was then added to react and quench with the extra oxidant. After adjusting the pH to 7 by adding 0.5 M HCl, the TEMPO-oxidized product was washed with D.I. water by centrifugation and further purified by dialysis against D.I. water for two days.

Carbohydrate-Binding Module Binding to OXCNC

[0157] Recombinant Resilin protein fused to cellulose binding domains was used to simulate the binding of CBM-containing allergens to OXCNC.

[0158] Binding was accomplished by incubating mixtures of res-CBD and CNCs at pH8 and pH 5.5 for 1 hours at room temperature, with gentle rotation of the sample. Mass ratios of 1:10 of res-CBD: CNCs were tested. After incubation, ultrafiltration was used to isolate unbound protein (EMD Millipore Centricon membranes, 0.2 μm pore size), since unbound res-CBD (MW≈53 KDa) passes through the membrane, whereas CNCs do not. The different mass ratio mixtures were centrifuged using a bench-top model (14000 rpm), and the permeates and retentates were collected and analyzed by SDS-PAGE to determine conditions for optimal binding. In addition, pure res-CBD was used as a control in order to establish non-specific interactions between the proteins and the membrane, while pure CNC was used in order to establish that CNC does not pass through the membrane. Equal amounts of protein (3.5 μg) from the total mixture (T), retentate (R), and permeate (P) of each of the tested mass ratios were loaded and separated on the acrylamide gel (12.5%), and compared to a protein ladder (7 μL, MW range 250-10 KDa) and to a sample of pure res-CBD (3.5 μg).

Resilin-CBD/CNC Binding Assay

[0159] Resilin-CBD/OXCNC in different weight ratios were incubated followed by protein separation via CENTRICON membrane with a CO of 0.22 μm. Permeats (˜100 μL) and Retentate (˜100 μL) were collected and analyzed by SDS-PAGE (12.5%). 1:10 wt. ratio of resilin-CBD/CNC is˜the maximum binding capacity using these conditions. Previous gravimetric tests indicated that unlike resilin-CBD, CNCs do not permeate the membrane to any measureable extent.

[0160] It was found by SDS-PAGE analysis that the optimal binding of res-CBD to OXCNCs under the conditions used in this work was pH8 (FIG. 1), at Ph5.5 about 50% of the protein binds to OXCNC (FIG. 2) while in pH 8, about 90% of the protein binds to OXCNC at weight ratios of 10:1. At lower weight ratios, we will probably obtain 100% protein binding to OXCNC.

Pollen Allergenic Proteins

[0161] Ambrosia pollen is notorious for causing allergic reactions in humans, specifically allergic rhinitis. Up to half of all cases of pollen-related allergic rhinitis in North America are caused by Ambrosia. (Taramarcaz, P.; et al. (2005). “Ragweed (Ambrosia) progression and its health risks: will Switzerland resist this invasion?” Swiss Medical Weekly. 135 (37/38): 538-48.)

Pollen Proteins Extraction

[0162] Native Ambrosia (A. confertiflora) pollen from wild plants suspended in PBS×1 buffer and incubated at room temperature for 20 min, and then centrifuged. The supernatants was separated from the pellet and used for allergen-binding assay to OXCNC and CNC.

Allergen-Binding Assay

[0163] Binding was accomplished by incubating mixtures of different mass ratios of soluble pollen proteins and OX CNC or CNC at pH7.5 for 1 hours at room temperature, with gentle rotation of the sample. Volumetric ratios of 1:3 and 1:10 of soluble pollen proteins: CNCs were tested. After incubation, ultrafiltration was used to isolate unbound protein (EMD Millipore Centricon membranes, 0.2 μm pore size), since unbound soluble pollen proteins passes through the membrane, whereas CNCs do not. The different Volumetric ratio mixtures were centrifuged using a bench-top model (10000 rpm), and the permeates and retentates were collected and analyzed by SDS-PAGE to determine conditions for optimal binding.

Binding of Pollen Allergenic Proteins to O×CNC and CNC

[0164] As FIG. 3 demonstrates, Ambrosia soluble pollen proteins passes easily through the membrane (CONTROL Ambrosia pollen proteins). Previous gravimetric tests indicated that unlike the soluble pollen proteins, CNCs or OXCNCs do not permeate the membrane to any measureable extent. Soluble pollen proteins that were incubated with OX CNC or CNC at pH7.5 bind to the CNC partial and remain with the OXCNC or CNC particles at the retentate in v:v ratio of 1:3 and 1:10.

[0165] Following the procedure in the microfluidizer, binding assays were performed using the same pollen as used to check the oxidized cellulose. Results show that the proteins in the pollen did indeed bind to the oxidized cellulose. Binding assays showing Lolium perenne pollen binding to different forms of cellulose, including oxidized cellulose and microcrystalline cellulose, oxidized cellulose and microfluidized CNC are shown in FIGS. 4A-C.