CROSS-REFERENCE TO RELATED APPLICATION APPLICATIONS

The present application is a national phase entry of PCT/SG2019/050045, filed on Jan. 29, 2019, which claims the benefit of priority of Singapore Patent Application No. 10201800742R filed on 29 Jan. 2018, the content of which is incorporated herein by reference for all purposes, including an incorporation of any element or part of the description, claims or drawings not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

The present invention relates generally to a new type of biomaterial that can be generated from pollen, methods for its production, the various uses thereof in, for example, biological, medicinal, cosmetic and nutritional applications and the materials/devices that comprise this new material.

BACKGROUND OF THE INVENTION

Pollen plays a critical role in plant reproduction, transferring cellular material between different reproductive parts of plants. Plants have evolved mechanisms to renewably produce pollen grains in large quantities, and the fidelity of these biosynthetic mechanisms is remarkably high. For each plant species, a particular type of pollen grain is produced that is composed of a hollow microcapsule structure with uniform size and function-driven shape and ornamental architecture. In all cases, the microcapsule structures possess a strong outer layer that is composed of sporopollenin, which is a crosslinked biopolymer comprising fatty acid, phenylpropanoid and phenolic components. Often regarded as the diamond of biopolymers, sporopollenin imparts extreme durability, mechanical strength and chemical stability to pollen grains. Such properties have led pollen to be regarded as practically indestructible and pollen is widely used as stable, content-rich markers for archaeological, ecological and forensics applications.

In nature, pollen shells have evolved not only to be the first defensive line in a quiescent, dehydrated state, to protect internal microgametophytes during indeterminate periods, but also to be a dynamic “smart” carrier in a responsive, rehydrated state, to transfer the haploid male genetic materials to a receptive stigma during pollination. Constitutive materials and nano-/microstructures of the pollen shell facilitate large volume changes of its pollen grain, particularly for harmomegathy, a self-defensive and environment-responsive process to maintain the internal moisture in arid environments by closing off the apertures of the shell. This phenomenon has long been mysterious until recently it has been evidenced as dynamic gelation of pollen grains, in which pectin acts like “valves” for water entry, enabling a regulated process of water uptake into the dehydrated pollen grain.

Sporopollenin-based sporoderms form the outer shell wall in a wide range of pollens (pollen grains) and plant spores so as to protect sensitive genetic material from harsh environmental conditions. The sporoderm polymer complex (SPC) typically comprises sporopollenin as an outermost layer (exospore or exine) with cellulose, hemicellulose, and pectin as an inner layer (so-called intine). The exine layer is often intricately sculptured in species-specific patterns, allowing material recovered from (for example) lake sediments to provide useful information to palynologists about plant and fungal populations in the past. The intine layer is located on the inside of the exine and adjacent to the pollen plasma membrane. The intine layer consists of two separate layers, the granular exintine with pectine and protein inclusions facing the exterior, and a microfibrillar cellulosic endintine facing the interior. The pectin network is known to interact with the cellulose network through both covalent ester linkages and non-covalent hydrogen bonding. Similar to pectinase, the treatment with strong alkali (potassium hydroxide; KOH) at high temperature (80° C.) de-esterifies pectin through an alkaline hydrolysis reaction, increasing water absorption capability due to the resultant gelation. Besides the influence on pectin, strong alkali not only disrupts hydrogen bonding between hemicellulose and cellulose microfibrils or between pectin and cellulose microfibrils, but also degrades pectin and cellulose molecules along with the increased duration of alkali treatment, ultimately leading to softening of intine. As a result, pollen transforms from the close state (or termed as “off”) to the open state (or termed as “on”) due to opening of apertures which are the weakest spots of the pollen surface, providing the main route for uptake of water and nutrients from the environments and serving as the exit point of a pollen tube.

There has been growing interest to explore pollen for materials science applications, including as templates for materials fabrication, structural reinforcements for composite materials and drug delivery vehicles among other possibilities. A key advantage is that pollen is readily obtainable from numerous plant sources at low cost and can be processed into isolated capsules. Defatted pollen grains, or more commonly extracted sporopollenin shells, are the main types of pollen-derived materials that have been used for microencapsulation and drug delivery applications.

However, the extremely high stability of sporopollenin has resulted in this remarkable material being largely overlooked in the fields of fundamental and applied materials science.

While it is known in the art to use acid/base processing strategies to produce pollen-derived capsules from pollen grains to remove sensitive biological materials such as proteins, polysaccharides and nucleic acids while preserving the physical and chemical properties of the sporopollenin shell, the present invention is based on the inventors' surprising identification of a one-pot processing strategy that challenges the prevailing notion that pollen is practically indestructible and structurally immutable. When pollen from sunflower plants (Helianthus annuus) was incubated in alkaline conditions for extended periods of time (a simple process replicating the basic step of soapmaking), it was discovered that the hard pollen grains were transformed into pliable, soft microgel particles. In marked contrast to the pollen precursors, the alkaline-treated microgel building blocks could be assembled into two-dimensional sheets and three-dimensional sponges, opening the door to an entirely new class of naturally sourced, bioinspired materials with advantageous properties. In addition, they could be directly applied to three-dimensional (3D) printing systems as either a “write-in” supporting matrix or a “write-out” smart material ink component.

To date, there have been no references to sporopollenin being dissolved, oxidized, and degraded/eroded under any chemical conditions except human fluid (e.g. specific enzyme or blood for partial degradation/erosion). Specifically, there have been no studies specifically exploring the base-hydrolysis degradation and gelation process of sporopollenin and no reports of strategies to design functional materials from pollen-based materials.

SUMMARY OF THE INVENTION

The present invention changes this by providing, in a first aspect, a method for the formation of microgels of sporoderm polymer complex microcapsules (SPC-MCs), and the method is comprised of the following steps:

(a) providing pollen grains from eudicot plants;

(b) deproteinizing the pollen by contacting it with an aqueous base solution at elevated temperatures for up to 10 hours to obtain porous SPC-MCs;

(c) hydrolytically degrading the SPC-MCs by contacting it with an aqueous base solution for periods up to 60 days to obtain microgels of SPC-MCs.

In these methods, the pollen may originate from a flowering plant of the genusBaccharis, HelianthusorCamellia, more preferablyHelianthus annuus.

In various embodiments of the inventive methods, the pollen used is either defatted pollen or the method further includes a step of defatting the pollen prior to step (b), optionally by treatment with an organic solvent, such as, without limitation, acetone. Irrespective of whether the pollen is defatted or not, it may be used in dry form for step (b).

The aqueous base solution used in step (b) and/or (c) may, in various embodiments of the invention, comprise, consist essentially of or consist of an aqueous alkaline metal hydroxide solution, preferably potassium hydroxide (KOH) or sodium hydroxide (NaOH) solution, more preferably KOH solution. In these solutions, the alkaline metal hydroxide concentration may range from about 0.5 to about 40% (w/v), preferably from 1 to 25% (w/v) relative to the total volume of the solution.

In various embodiments, step (b) is carried out at a temperature of 40 to 95° C., preferably 60 to 90° C., more preferably 70 to 85° C. and/or for a period of 1 to 6 hours, preferably 2 to 4 hours.

Step (c) may be carried out at a temperature of 10 to 30° C., preferably 15 to 25° C., and/or wherein step (c) is carried out for 7 to 35 days, preferably 14 to 28 days.

In various embodiments, step (c) further includes a step of neutralizing the suspension of partially degraded SPC-MCs after the hydrolytic degradation by washing the partially degraded SPC-MCs with water until the suspension has a pH in the range of 6.0 to 8.5, preferably in the range of 6.5 to 8.0, more preferably in the range of 7.0 to 7.5, to obtain a neutralized microgel solution.

The microgel obtained may typically be a viscous aqueous gel comprising 1 to 6 wt.-% SPC-MCs and the remainder being water.

In a second aspect, the invention also encompasses a microgel comprising SPC-MCs obtainable by a method according to the invention.

In a still further aspect, the invention features a method for the formation of flexible sheets or sponge structures, comprising the steps of forming microgels of sporoderm polymer complex microcapsules (SPC-MCs) according to the method of the invention and further comprising the steps of

(d) forming a film of the microgel on a substrate and drying the film to obtain a flexible sheet of SPC-MCs; or

(e) lyophilizing the microgel to obtain sponge structures of SPC-MCs.

Flexible sheets or sponges comprising SPC-MCs obtainable by the method described herein also form part of the invention.

Another aspect is directed to a composition comprising the microgel or the flexible sheets or sponges according to the invention, wherein the composition is a cosmetic, nutritional or pharmaceutical composition. Alternatively, the composition may be a printing composition, such as a composition for 3D printing or a printing ink composition.

The microgels of the invention may, for example, be used as a thickener or gelling agent for cosmetic or food compositions. Also contemplated is their use in 3D printing and ink compositions. Alternatively, the microgels, flexible sheets or sponges may be used as scaffolds for cells, for example in cell culture or medicinal applications, for example to stimulate cell proliferation.

A still further use of the microgel, the flexible sheets or sponges is in the delivery of an active agent, wherein said active agent is preferably encapsulated in the SPC-MCs. The active agent may be a pharmaceutically or cosmetically active agent or a cosmetic or pharmaceutical composition.

A still further aspect relates to a method for promoting cell proliferation comprising

(a) contacting cells with a sheet or sponge according to the invention; and

(b) culturing said cells in the presence of said sheet of sponge as a scaffold under suitable culturing conditions.

Further encompassed is a method for the delivery of a pharmaceutically or cosmetically active agent, comprising providing a composition comprising the microgel or the flexible sheets or sponges of the invention, the microgel, flexible sheets or sponges comprising the pharmaceutically or cosmetically active agent encapsulated in the SPC-MCs, and administering a pharmaceutically or cosmetically active amount of the composition to a subject.

In the following the invention will be described in greater detail by reference to the accompanying drawings and examples.

