WO2018033744A1 - Carrier - Google Patents

Abstract

The present invention relates to a mucoadhesive carrier or drug delivery system, which comprises nanoscaffold having a nanofibrous layer with a thickness of from 0.1 to 1000 µm, carrying the active substance in the form of particles. The mucoadhesive layer, in at least a part of its surface, overlaps the nanoscaffold so that it can adhere to the mucosa. A process for its preparation (the nanofibres are formed by electrospinning) and its use for delivery of the vaccines and therapeutics to mucosal surfaces is also disclosed.

Description

CARRIER

Field of Art The present invention relates to a (drug or active ingredient) delivery system, in particular a mucoadhesive reservoir carrier (or patch), particularly suitable for, or capable of, carrying and/or administering an (active) substance (such as in the form of particles) to a (e.g. sublingual) mucosa of a human and/or animal. Background Art

Particulate carriers of vaccines, drugs and other physiologically active substances (e.g., plasmid DNA, siRNA, therapeutic peptides and proteins, antigens, allergens) are used in the treatment and prophylaxis of a number of diseases in humans and animals. Formulations based on nanoparticles and microparticles are usually administered orally and parenterally. The administration of microparticles and nanoparticles to different types of mucous membranes can be non-invasive and painless, with rapid absorption, minimized risk of infection, and bypassing of the digestive system and the portal blood circulation (De Jong WH, Borm PJ, Int. J. Nanomedicine. 2008; 3(2): 133-149., Micro- and nanoparticles— medical applications, Jatariu A, Peptu C, Popa M, Indrei A, Rev. Med. Chir. Soc. Med. Nat. Iasi. 2009 Oct-Dec; 113(4): 1160-9).

The total surface area of the oral mucosal lining in a human is approximately 100 cm². The oral mucosa can be divided into the following 3 types: buccal mucosa, sublingual mucosa and palatal mucosa. Individual types of mucosa anatomically can vary in their thickness, degree of the epithelium keratinization, and hence the permeability for drugs, particles and other physiologically active substances. These mucosal categories also differ significantly in the structure (or proportions of the immune cell types). In humans, the sublingual mucosa is the thinnest, without signs of keratinization, whereas buccal mucosa is thicker, but also without signs of keratinization. The palatal mucosa is the thickest one and is keratinized and hence the least permeable for drugs and particles. In general, the oral mucosa consists of multiple layers, namely a layer of epithelium whose cells flatten towards the surface; basal membrane; lamina propria layer; and submucosal tissue which receives a blood supply and contains numerous nerve endings. The upper layers of the epithelium contain materials of lipophilic nature and intracellular origin stretching between cells, forming a barrier to the passage of particles and substances through the mucosa (Gandhi RB, Robinson JR, Adv. Drug Deliv. Rev., 1994; 13: 43-74).

The main barriers blocking the passage of particles and drugs into, and across, the oral mucosa are (1) the mucus layer on the mucosal surface (Cone RA. Adv. Drug Deliv. Rev., 2009; 61: 75-85), (2) the keratin layer (where present), (3) intercellular lipids of the epithelium (Chen LL, Chetty DJ,Chien YW.Int. J. Pharm. 1999; 184: 63-72), (4) basement membrane and (5) an enzymatic barrier (Madhav NVS, Shakya AK, Shakya P, Singh K. J. Control. Release. 2009; 140: 2-11). Significant external factors influencing the penetration of particles through the mucosa include continuous production of saliva (so washing the mucosal surface and forming a thin film), and movement of the oral mucosa and tongue during speaking, eating, drinking and chewing. Given the similarity of structure and degree of keratinization of mucosa with humans, the pig is currently the most widely used model animal for monitoring the transfer of substances and particles through the oral mucosa (both in-vivo and ex-vivo experiments).

The effectiveness of some particulate carriers of mucosal vaccines fail because of their lack of effect on the immune cells and the immune system, or insufficient penetration of these particles across the mucosa, especially in model animal species.

Given the barriers and physiological conditions in the oral cavity, the active substances need to be specially prepared and administered by appropriate administration forms. The standard oral drug forms include buccal and sublingual tablets, pastilles, sublingual sprays, oral gels and solutions. However, these drug forms do not allow the ingestion of food or drink, and in the case of sublingual sprays even during speaking. These formulations are preferred for dealing with the administration of low-molecular substances and insulin. More advanced mucoadhesive drug forms can include solutions (which form a viscous gel directly on the mucosa), sublingual effervescent tablets and mucoadhesive buccal and sublingual films.

Prior art drug delivery systems often have insufficient active ingredient, for example, the loading or concentration may not be high enough and/or they are unable to deliver (or release) sufficient active ingredient to the site of interest (normally the mucosa). There is therefore a need for a mucosal delivery system that has a drug loading, or concentration of active ingredient, that allows the drug or active ingredient to be delivered quickly and effectively to the desired site (usually the mucosa). The invention seeks to solve at least one of those (prior art) problems, or at least mitigate these drawbacks of prior art drug delivery systems.

Disclosure of the Invention

A first aspect of the present invention provides an (improved) drug (or active ingredient) delivery system (e.g. reservoir or patch) which can be a (muco-adhesive) carrier, composition or formulation comprising a nanoscaffold. This may be a sustained or cotrolled release system. The nanoscaffold preferably comprises a (nano)fibrous layer and/or (nano)fibres: suitably it may carry, or comprise, at least one (e.g. active) substance (i.e. drug, API or a mixture of substances) preferably comprising, or in the form of, particles. It may thus act as a reservoir or depot for the drug/ API. The mucoadhesive layer preferably, over at least part of its surface, overlaps the nanoscaffold. The carrier can be adapted so that (during its use) the nanoscaffold faces or contacts the mucosa and/or the mucoadhesive layer serves to (or is capable of) attach or adhere the carrier to the mucosa. The system may have a high drug loading or concentration of the (active) substance.

The invention also relates to a mucoadhesive (drug or API) delivery system comprising:

a) a matrix (e.g. a nanoscaffold) comprising at least one active substance or pharmaceutical ingredient (API); and

b) a mucoadhesive (or mucoadhesive means) adapted to adhere, or capable of adhering, the system to a mucosa. The invention may be for medical, therapeutic, diagnostic, vaccine use, such as local/synthetic (gene) therapy, sublingual delivery (immuno) therapy or vaccination.

Suitably:

a) the matrix comprises a nanoscaffold (and/or one or more biocompatible polymers) and/or has pores of size from 10 nm to 100 μιη;

b) the mucoadhesive (layer) comprises a portion suitably capable of, or adapted to, secure, attach or adhere the system (or matrix or nanoscaffold) to a mucosa or mucosal surface;

c) the mucoadhesive layer (at least in one part thereof) overlaps or is larger (in surface area) than the matrix, and/or the sytem has an exposed part of the (muco-adhesive) layer e.g.

(facing) towards the mucosa; and/or

d) the API (drug, active substrance, pharmaceutical, vaccine) is in the form of, or comprises, particles. Mucoadhesive

The mucoadhesive part or portion (normally meaning adapted or suitable for adherence, or capable of adhering to, or contact, with a mucosa) can be a layer. At least part of its surface may overlap the nanoscaffold. Thus part of (the surface of) the mucoadhesive (layer) may extend (or overlap) beyond an edge of the nanoscaffold. This (overlapping or exposed) part of the surface of the mucoadhesive layer (e.g. extending beyond the nanoscaffold) can (serve to) attach, or be capable of attaching, the carrier to the mucosa, such that the nanoscaffold may be adjacent or adhered to the mucosa. The whole structure/system may thus be fixed onto the mucosa by the adherence of part of (the surface of) the mucoadhesive layer or portion, suitably extending beyond (or overlapping) the nanoscaffold. In this sense the mucoadhesive (part or portion of the system) can have a larger (surface) area than the nanocaffold (or matrix). The muco-adhesive may be in the form of or comprise a layer, and/or may be adjacent to the nanoscaffold (or separated therefrom by an interlayer or intermediate layer). The mucoadhesive may be adhesive on both sides, for example of the mucoadhesive layer, or only on one side. The muco-adhesive may have a cover layer e.g. substantially covering all of one side of the muco-adhesive layer. Although the muco-adhesive may be in the form of a layer, it may also be in the form of a number of different sections and/or portions, some or each of which may be muco-adhesive. For example, the muco-adhesive may be in the form of spots and/or dots, e.g. on a layer in the system or carrier or it may be flush and/or adjacent to the nanoscaffold layer. There may be several areas where the portions of the muco-adhesive are useful for adhering to the mucosa. If, for example, the carrier or system is circular, or indeed another geometrical shape, the muco-adhesive layer may be part of or comprise the perimeter or surrounding area (such as in the form of a ring or annulus) and/or may have a central (e.g. circular) portion for adherence. The muco-adhesive layer may therefore occupy and/or comprise the circumference of the carrier, and this may be in a single continuous section or may be in a series of one or more smaller sections (such as spots or dots), for example in a (circular) ring or annular portion of the system. APIs (therapeutic, diagnostic, prophylactic agents, etc.)

The substance (e.g. in the form of particles) can comprise the active substance (or API) itself which is suitably carried by, or present in, the carrier. It can be transported to the target mucosa, and/or transmucosally, for example to reach draining lymph nodes and/or systemic circulation. Preferably the API is in the form of, or comprised in, particles (the active substance itself may be capable of forming particles) and/or is with at least one carrier and/or an excipient, which together may form a particle comprising the active substance (or API). There may be a mixture of active substances (APIs), usually intended for delivery to the target mucosa, optionally with at least one carrier and/or excipient, which together may form or comprise a particle (containing the mixture of active substances).

Nanoscaffold (or matrix)

The nanoscaffold (or matrix, the terms are used interchangeably) may be a three-dimensional structure, e.g. a layer. It can be formed by, or comprise, a (layer of) biocompatible polymer(s) or a mixture thereof. Suitably the nanoscaffold comprises one or more nanofibre(s), thus providing its structure. This may provide space for the API, such as to be absorbed or located or adhered therein. It may also allow the API to leach, wash out, dissipate, be released from or exit from the nanoscaffold, such as over time, e.g. in a sustained or prolonged release mechanism. The nanoscaffold may thus allow easy entry (loading) and/or exit (release) of the API to and/or therefrom.

The (nano)fibre(s) suitably have a length in the range of centimeters to several meters. They suitably have a thickness (diameter) of from tens of nanometers to tens of microns (e.g. from 1, 5 or lOnm to 50, 100, 150, 250 or 500nm, e.g. from 10 to 150nm). The nanofiber(s) may thus provide a large (internal) surface area, and/or have a large interfibrous (pore) volume within the nanoscaffold. Preferably the particle(s) adhere to, or are in contact with, the nanofiber(s).

The nanofibrous layer (preferably with a thickness in the range of 0.1, 1 or 10 to 100, 500 or 5,000 μπι, preferably 1 to 50 μπι, suitably 100 nm to 1 μπι) can thus comprise a layer of nanofibres. It can be formed of nanofibres, e.g. comprising biocompatible polymers (or a mixture thereof), preferably with a thickness in the range of 10 or 50 nm to 1, 10 or ΙΟΟμπι, preferably 50 to lOOOnm. They may form or comprise a net or scaffold and/or mesh (structure). This suitably does not (substantially) sterically hinder the movement of the (carried) nanoparticles and/or microparticles therefrom or therethrough.

The nanoscaffold may contain pores with sizes ranging from tens of nanometers to hundreds of micrometers (e.g. 10 or 100 nm to 100 μπι, preferably 0.1 or 1.0 to 10 or 100 μπι). The nanoscaffold (layer) may have or comprise a nanofibrous structure, foam structure, or (structure of) plates, crystals or other shapes. It may be from about 100 nm to 3mm thick (deep), preferably 1 - 50 μπι.

The nanofibres or nanoscaffold may comprise one or more polymers such as polyamides, polyurethanes, polyethersulphones, polyvinyl alcohol, polyvinyl butyral, polyacrylonitrile, polyethyleneoxide, polystyrene, polyvinylidene fluoride, polyvinylpyrrolidone, povidone- iodine, alginate, (silk) fibroin, polyacrylic acid, polyglycolic acid, polyacrylic acid, gelatine, chitosan, collagen, polyaramid, polylactic acid, poly-s-caprolactone, hyaluronic acid and/or (supersaturated) collagen. The surface of the nanofibers can be (further) physically or chemically modified, such as for the purpose of binding and/or release of particles, in particular macromolecular particles (e.g. proteins, DNA/RNA, polysaccharides) and/or nanoparticles or microparticles of low- molecular substances.

Examples of suitable modifications are: change in surface charge and its density, change in surface wettability rate, attachment of ligand(s) for selective binding, such as metallochelating complexes, specific ligands - biotin, monoclonal antibodies and their fragments, peptides, etc.

The surface of the nanofibres can be modified by (introduction of various) functional groups (e.g. amino-oxy, thio, carboxy, amino, azido, amido, hydroxyl, guanidino, benzyl, phenyl, indole, saturated and unsaturated lipids) such as for electrostatic or covalent (reversible or irreversible) binding of particles (onto the surface of the nanofibres).

The fibres may preferably be made by electrospinning or are electrospun. They may comprise natural and/or synthetic fibre(s) (also see the scheme in Fig 25).

Mucoadhesive component(s)

The mucoadhesive layer usually comprises one or more biocompatible substance(s) or a mixture thereof. It may have the ability to attach, or adhere, to a mucosal surface, e.g. due to interactions with the mucus layer (present on mucosal surface). The layer may comprise: polyacrylate (carbomers, Carbopol, polycarbophil), cyanoacrylate, tragacanth, xanthan gum, hyaluronic acid, guar gum, gelatine, pectin, polyvinylpyrrolidone, polyethylene oxide, sodium alginate, chitosan, dextran, cellulose derivative (e.g. hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, oxycellulose), poloxamer, copolymer of acrylic and methacrylic acid (Eudragit), lectin, thiolated polymer - thiomer (e.g. chitosan-N-acetylcysteine, chitosan- cysteine, chitosan-thioglycolic acid and/or carboxymethyl cellulose-cysteine, alginate- cysteine). The mucoadhesive layer may further contain one or more plasticizers, e.g. a substance that may provide deformability and/or plasticity, e.g. of the layer (comprising, for example, glycerol, polyethylene glycol, propylene glycol), phthalate (e.g. dibutyl phthalate), citrate (e.g. triethylcitrate) or a surfactant (sodium lauryl sulphate, sodium deoxycholate, sodium cholate, triton and the like).

The mucoadhesive layer and/or the nanoscaffold (matrix) may also contain one or more of:

(a) excipient(s) that may facilitate the penetration of particles into the mucosa;

(b) a substance able to decrease the mucus layer viscosity (a mucolytic, e.g. acetylcysteine); and/or

(c) a surface-active substance (sodium deoxycholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurocholate, taurodeoxycholate, sodium cholate, sodium lauryl sulfate, polysorbates (TWEEN80), polyoxyethylene, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride, etc.); and/or

(d) a chelating agent (e.g. ethylenediaminetetraacetic acid, EDTA) and/or fatty acid (e.g. oleic acid, capric acid, lauric acid, methyl oleate) and/or polyols (e.g. propylene glycol, polyethylene glycol) and/or dextran sulphate and/or sulfoxides (e.g. dimethyl sulfoxide), and/or Azone® (l-dodecylazacycloheptan-2-one), phosphatidylcholine, lysophosphatidylcholine, methoxysalicylate, menthol, aprotinin, dextran sulphate, cyclodextrins or 23 -lauryl ether.

The mucoadhesive layer and/or the nanoscaffold may also contain an inhibitor of a proteolytic enzyme. The shape of the mucoadhesive carrier (and/or system/carrier) is preferably (substantially) circular, eliptical or oval (or section of any oval form), reniform, trigonal, tetragonal or polygonal shape. The size of the mucoadhesive carrier can be adapted to the physiological dimension of the recipient (human, animal). Generally the area of nanoscaffold is in the range of 1 - 10,000 mm² Cover layer (s)

The carri er/sy stem may further comprise a cover layer. This may be instead of, or in addition to, a mucoadhesive layer. In the former case the cover layer may carry, contain or comprise the mucoadhesive. Thus the mucoadhesive may be (a) optionally, a cover layer; located on a cover layer (e.g. see Figure 1, CC and DD).

The sequence of layers is preferably:

(a) optionally a cover layer;

(b) mucoadhesive layer (e.g. overlapping the nanoscaffold in or on at least part of its surface);

(c) optionally an inter-(vening) or an intermediate layer; and/or

(d) a muco-adhesive layer.

The mucoadhesive layer may be adjacent to or connected (or in contact with) the cover layer (in part of its surface) and/or the nanoscaffold can be connected/in contact with the cover layer (over part of its surface). The mucoadhesive layer may thus be located between, or sandwiched by, the cover (protecting) layer and nanoscaffold.

The cover layer (in itself) may not be mucoadhesive. It may be inert, non-porous and/or impermeable (e.g. to the API and/or particles). It may comprise a film-forming substance or a substance which has or can be spun. The substance can be used either alone or in a mixture with other substances mentioned above and/or substances regulating the layer properties (plasticizers, surfactants, agents adjusting pH, ionic strength, etc.). Examples of suitable substances which can be used in, or to comprise, the cover (or intermediate) layer are on or more of cellulose derivatives (ethyl cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, oxycellulose and cellulose acetate phthalate, celacephate), copolymers of esters of acrylic and methacrylic acids (Eudragit^®), polyacrylates (carbomers, carbopol, polycarbophil), cyanoacrylates, hyaluronic acid, gelatine, pectin, polyvinylpyrrolidone, polyethylene oxide, alginates, gum arabic, shellac, chitosan, wax, stearic acid, dextran, poloxamer and/or polycaprolactone. A polyol (glycerol, polyethylene glycol, propylene glycol), phthalate (e.g. dibutyl phthalate) and/or citrate (e.g. triethyl citrate) can be included, e.g. used as a plasticizer. The thickness of the cover (or intermediate) layer may be variable, preferably from 0.1 or 1 μιη to 100 or 200μιη. It can be arranged in the form of (or comprise) a polymer film or nanofibers.

This layer can block, or prevent, the penetration or movement of particles and/or molecules (e.g. in a direction away from the mucosa). It may ensure a (high) local concentration (of particles and molecules) is delivered to the mucosa, suitably for a sufficiently long period (time interval of the order of (tens of) minutes to hours).

The cover (or inter) layer can be deposited or made, for example, by spraying or (electrostatic) spinning a polymer solution (e.g. on the mucoadhesive layer). Suitably the cover layer can prevent the system adhesion to the applicator (or to a finger) such as during the administration process. It may supply or assist a required mechanical property to the (entire) system: it may ensure easy handling of the formulation and/or after application prevent mucoadhesion to a site other than the intended site of administration. It may extend the adhesion interval and/or prevent the release of (nano)particles from the nanoscaffold (e.g. into the oral cavity).

The cover (or inter) layer can be (entirely or substantially) insoluble and/or can gradually dissolve in use. An (entirely) insoluble layer may extend the (interval of the) carrier adhesion and/or prevent leakage of particles during the period the carrier is at the site (of administration). In a preferred embodiment, the cover layer comprises one or more soluble materials, and these may dissolve at a rate such that dissolution of the individual layer(s) is avoided, or reduced, such as before the particles (e.g. API) are released from the carrier.

After the release of the particles and subsequent disintegration of the carrier (e.g. by an erosion mechanism), and/or dissolving of different components, it may not be necessary to remove the carrier. In the case of incomplete dissolution (after the required administration interval) fragment(s) may get moved to other parts of the digestive system together with saliva, or with food or drink. This may not be harmful because suitably all materials used in the carrier are inert and/or biodegradable (in the digestive tract).

Intermediate layer

The mucoadhesive carrier, or system, preferably comprises an intermediate layer. This may be adjacent to, or in contact with, the nanoscaffold. The intermediate layer may be on the side of the nanoscaffold which is not adjacent to, or away from, the mucosa. It may be located next to, or in between, the mucoadhesive layer and the matrix/nanoscaffold.

The intermediate layer may comprise a polymer, or another substance, suitably without (or substantially no) mucoadhesive properties. Its thickness can be variable, preferably from 0.1, 1 or 50 μπι to 100, 200 or 300μιη. It can be arranged in the form of, or comprise, a polymer (film) or nanofibers. It can be placed or located between the nanoscaffold/matrix and/or the mucoadhesive layer and/or cover layer (and it may contact one or both).

The intermediate layer can be impermeable, such as to the API or particles, e.g. carried or present in the nanoscaffold. This may prevent, or reduce, their washing out, movement or exit from the carrier (e.g. in the direction away from the mucosa). The (insoluble or sparingly soluble) intermediate layer is preferably prepared from, or comprise, a polymeric film- forming substance, e.g. commonly used in pharmaceutical technology (or is prepared from or comprise spun polymers arranged into nanofibers).

The intermediate layer may comprise one or more of: a cellulose derivative (ethyl cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, oxycellulose, and cellulose acetate phthalate, celacefat), copolymer of esters of acrylic and methacrylic acids (Eudragit^®), polyacrylate (carbomer, Carbopol, polycarbophil), cyanoacrylate, hyaluronic acid, gelatine, pectin, polyvinylpyrrolidone, polyethylene oxide, alginates, gum arabic, shellac, chitosan, wax, stearic acid, dextran, poloxamer and/or polycaprolactone. It may (also) comprise one or more plasticizers, for example polyol (glycerol, polyethylene glycol, propylene glycol), a phthalate (e.g. dibutyl phthalate) and/or citrate (e.g. triethyl citrate). The intermediate layer may prevent, or reduce, (particle) leakage or movement from the nanoscaffold into and/or through the mucoadhesive layer. This leakage, or movement, may occur due to swelling of the mucoadhesive polymer, e.g. due to osmotic forces and/or movement or diffusion of particles and/or the API. The individual layers should ideally be prepared in advance and (firmly) attached to, or in contact, with each other. They may be deposited in the form of a spray (of a solution) of a layer-forming substance, e.g. solid particles of a layer-forming substance or a layer-forming substance (in the form of nanofibres). The attachment may occur simultaneously with the formation of the layer.

API and other (active) substances or ingredients

The substance (API) may be in the form of, located in or comprise particles. These may be incorporated into, or located in, the nanoscaffold, e.g. after its formation (they may not be part of the nanofibres (or its structure) but may be located within the nanoscaffold itself). Thus they may be anchored (e.g., by (reversible or irreversible covalent) binding or non-covalent interactions) located, imbedded or absorbed there. The particles can be (or comprise) liposomes, nanoparticles, lipid-based nanoparticles (LNPs), polymer-based nanoparticles (PNPs), microparticles and/or macromolecules. They may have (or comprise) mucosa (mucus) penetrating properties and/or be mucosa (mucus) penetrating particles.

Particles

The nanoparticles can have a size range of from 1 or lOnm to 500, 1,000 or 5,000 nm. Suitably they comprise a biocompatible substance. The substance may comprise an aliphatic polyester (polylactic acid, polyglycolic acid or copolymer of lactic and glycolic acids, poly-ε- caprolactone), polyalkyl cyanoacrylate, polyhydroxyalkanoate, hydroxymethyl methacrylate, polystyrene sulfonic acid, polystyrene-poly(ethylene glycol), poly(organophosphazene), polyethylene oxide, gelatine and/or polysaccharide (chitosan, hyaluronic acid or alginic acid).

Lipids and phospholipids are often used in the formulation of liposomes or lipid-based nanoparticles (LNPs). They may comprise liposomes, solid lipid nanoparticles (SLN), bilosomes, virosomes and virus like particles, polymeric nanoparticles and/or lipoplexes.

The particles may carry (as examples of an API) or comprise a drug, antigen, allergen, vaccine, physiologically active substance, nucleic acid, protein, peptide and/or polysaccharide. The particles may comprise any of the listed agents (e.g. drug, antigen, immunogen protein, polysaccharide, nucleic acid, therapeutic antibody), or for example viruses, virus-like particles, LNPs, polymer particles or (solid) lipid particles or hybrid polymer-lipid particles. Suitable particles comprise: liposome, polymeric nanoparticle, dendrimer, niosome, conjugate of low-molecular substance and polymer, complexes of a substance with cyclodextrin, nanoemulsion or bacterial envelope. The nanoparticles can comprise micelles (prepared from surfactants or their mixtures). They may be embedded within (e.g. not a component of the nanofibers) the (nano)scaffold mesh (or matrix).

The particles can be loaded into or located in the nanoscaffold (reservoir) layer by any suitable process such as spraying, sputtering, dropping and/or printing, such as of a nanoparticle preparation (homogenous particle preparation or a mixture) e.g. onto or into nanoscaffold reservoir layer, or by immersion of the nanoscaffold (reservoir) layer into a solution, or dispersion, of the particles (or API).

(Micro)particles can comprise particles of a size from 1, 2 or 5 μιη up to 10, 20 or 50 μιη.

The particles may comprise a (biocompatible) substance, so can be suitable for the preparation of microparticles. They may carry, contain or comprise a drug, antigen, allergen, physiologically active substance, nucleic acid, protein, peptide and/or a polysaccharide. The (nano)particles can be formed by, or comprise, any of the above mentioned substances (e.g. drug, antigen, protein, polysaccharide, nucleic acid) or may comprise of bacteria, or other pathogens or their fragments.

The (micro)particles may comprise an aliphatic polyester (polylactic acid, polyglycolic acid and their copolymers, poly-s-caprolactone), polyalkyl cyanoacrylate, polyhydroxyalkanoate, hydroxymethyl methacrylate, polystyrene sulfonic acid, polystyrene-poly(ethylene glycol), poly(organofosfazen), polyethylene oxide, gelatine and/or polysaccharide (chitosan, hyaluronic acid, alginic acid). Lipids and phospholipids can also be used in formulation of liposomes.

The particles may be modified in order to provide them with, or increase their, ability to penetrate the (mucus) layer, e.g. without significantly reducing the speed of their diffusion movement (regarding the speed of the diffusion movement of particles in an aqueous medium having a viscosity close to water). This can be achieved by modification of the particle surface using polyethylene glycol, or another hydrophilic electroneutral polymer, which may impart a surface charge close to zero to particles and their surfaces have a hydrophilic character (Frohlich E., Roblegg E.J. NanoSci. Nanotechnol. 2014 Jan; 14 (1): 126-36).

Suitably the particles have little or no mucoadhesive or bioadhesive properties. They can be bound, anchored, absorbed, located or embedded in spaces, voids, gaps, interspace(s) or interstation(s) in or inbetween (the (nano)fibres) of the nanoscaffold.

Other component(s) Besides the active substance and/or API, the matrix/nanoscaffold or system may also optionally contain absorption accelerator(s) and/or excipient(s), e.g. an excipient facilitating the release of particles carried to the mucosal surface and/or penetration of the particles through the mucus layer and/or penetration of the particles into the mucosa. An absorption accelerator (e.g. acetylcysteine), may be included, as e.g. at the site of administration, it may loosen the structure of the adjacent mucus layer and/or loosen the intercellular structure of the epithelium, particularly of the extracellular lipids contained in the upper third of the epithelium.

Excipient(s) may further include, for example, a cryoprotectant, antioxidant, stabilizer, antimicrobial agent, surfactant, e.g. detergent, tenside, emulsifier, mucolytic, sucrose and/or deoxycholate.

A cryoprotective agent, when included, may ensure the maintenance of particle stability during the lyophilisation process. Formulation of particles and nanoparticles into a mucoadhesive carrier may allow one to combine a variety of substances necessary for the functionality and stability of the components during the manufacturing process and the product storage.

The nanoscaffold may serve as a reservoir of the (micro)particles or (nano)particles. These may be reversibly (physically or chemically) adsorbed into, or on, the nanofibers and/or are (freely) distributed among the drug/ API release (nano)fibres. Particles can be (spontaneously) released from the nanoscaffold e.g. after administration of or adherence /contact the mucoadhesive carrier to the mucosa. The particles or API may be released by dissolution or erosion of the (nano) fibres of the nanoscaffold. The particles may also be released by diffusion or by osmotic force from the non-soluble nanoscaffold. Particles can serve as a reservoir of the drugs/API and can be bound onto the surface of nanofibres by stabile bonds (e.g. covalent chemical bonds or affinity bonds).

Drug/API release

The nanoscaffold, e.g. serving as a reservoir of particles, can have (an appropriate size of) pores in the structure and/or mesh(es) between (individual) nanofibres, suitably which may not impede or reduce the diffusion movement of the (carried) particles. The advantage is that the viscosity (of the solution) inside the nanoscaffold in which the particles move may be unaffected by the carrier properties. This problem can be encountered in prior existing systems which use mucoadhesive gels of high intrinsic viscosity. In order to release the particles from the gel layer, it is, first of all, necessary to hydrate the gel and disintegrate its structure, which reduces the transmission efficiency of the carried particles to the mucosa. The rate of the diffusion movement of particles in the nanoscaffold is only dependent on the viscosity of the outer aqueous environment. In the invention the (very) large or high surface area of nanofibers or pores in a matrix can provide a high capacity for adsorption of the particles. Simultaneously, a large amount of space may be available for depositing or locating the nanoparticles therein or therebetween. The extent and rate of API/particle release (from the mucoadhesive particle carrier) may be influenced by both (surface) properties of the nanofibres and/or the pores in the nanoscaffold and/or surface properties of the (carried) particles. These properties may include the hydrophilic/hydrophobic character of the surface of nanoparticles and nanofibers or pores, surface charge of nanoparticles and nanofibers or pores, shape and size of particles, and structure of the carrying nanofibres or pores. The rate and extent of release of nanoparticles from the nanoscaffold can be increased by surface modification of the nanofibers or pores (e.g. by increasing the rate of wettability by surface oxidation of nanofibres or pores in the plasma, by treatment of the nanoscaffold with a sodium hydroxide solution, or by adsorption of suitable surfactants (such as bile salts, sodium lauryl sulphate, and others), and also by surface modification of nanoparticles, for example by changing or influencing the particle charge, or preferably by surface modification of particles (e.g. with polyethylene glycol). The surface of the particles can be modified by adsorption of e.g. a surfactant.

Manufacture

The invention also provides a process for the preparation of the mucoadhesive carrier (or system), wherein a nanoscaffold may be prepared, subsequently attached to or contacted with a mucoadhesive layer and/or a cover layer. In a preferred embodiment, prior to the attachment or contact, an intermediate layer can be incorporated or located between the nanoscaffold and the mucoadhesive and/or the cover layer. In one preferred embodiment, the mucoadhesive and/or the cover layer and/or the intermediate layer will be formed, for example, by spraying a (polymer) solution, and optionally drying the solvent or by polymer solution casting and drying (solvent casting method). In another preferred embodiment, the mucoadhesive layer and/or the intermediate layer and/or the cover layer will be made in the form of nanofibres (e.g., by electrostatic spinning or electrospinning), and then (firmly) attached in the desired order. In another embodiment of the method, the nanoscaffold is prepared in situ on the mucoadhesive and/or the cover layer and/or the intermediate layer. If the nanoscaffold comprises a nanofibrous layer, it can be prepared, for example, by electrostatic spinning.

A substance, preferably in the form of a solution, colloid, or suspension can be deposited or located on or within the nanoscaffold, either after its production or after addition or completion of all other layers of the mucoadhesive carrier. In a preferred embodiment, the mucoadhesive carrier with the carried substance can be subsequently lyophilized or dried. This may enable problem-free long-term storage, important in the case of e.g. vaccines.

Suitably the drug or API is incorporated or located in or within the nanoscaffold and/or matrix, usually after the nanoscaffold and/or matrix has been formed. In other words, it is preferable that the drug or API is not incorporated or located into the matrix of nanoscaffolding when the latter is being prepared. So, for example, if the nanoscaffold is being prepared by electrospinning, it is preferred that the nanoscaffold is prepared first (e.g. using an electrospinning method), and then at a later time, or afterwards, the drug and/or API is incorporated into or located in the nanoscaffold. Other aspects

The invention further provides a (non-invasive) method of administration of a substance (API), e.g. in the form of particles, to a mucosa. The invention envisages sublingual, buccal, oral and/or vaginal mucosa. The mucoadhesive carrier can be delivered or contacted directly to the target mucosa either manually or by using a device, e.g. by simply applying or pressing for 1 to 30 seconds, preferably 3 to 10 seconds. The nanoscaffold is usually turned towards or (placed) in contact with the mucosa. After releasing the pressure, the carrier can adhere to or attach to the mucosal surface (e.g. due to the mucoadhesive forces arising between the mucoadhesive layer and the layer of mucus on the mucosa).

Compared with commonly used methods and carriers (especially the delivery of particles with mucoadhesive properties), this method of (non-invasive) administration of a carrier may achieve a high local concentration of nanoparticles/microparticles in a close proximity to the mucosal surface for enough time to achieve the required effect of the active substance. These factors can allow a more effective transfer of particles to a mucosa, thus allowing the induction of a therapeutic or prophylactic effect. The administered total dose of particles and substances carried by the particles can be lower. The influence of the flow of saliva, the movement of mucosa and tongue to remove particles from the mucosa during common activities such as eating, drinking and speaking may be eliminated or reduced. The effect of dilution of the administered particles with the ingested fluids can be considerably reduced. The invention may solve or ameliorate the problem of providing uniform dosage of particles and substances carried by the particles, since the mechanisms of particle elimination from the mucosal surface can be considerably suppressed. The proposed solution may eliminate the drawbacks of prior existing delivery systems to mucosal surfaces. These prior systems are based on the delivery of particles with mucoadhesive surface modification, which in turn can adversely affect the penetration of particles to the mucosal surface due to interactions with mucus. Thus, although the particle may remain at the delivery site where the substance can be released, it may be unable to effectively penetrate into the mucosa via the mucus layer. In the prior art, mucus-penetrating particles can be administered, but they (on the contrary) do not reside in the delivery site for a long period of time because they can be removed by movement of the tongue and fluids present in the oral cavity, and so are carried to other parts of the digestive system. The precondition for dosage uniformity, as well as of other oral dosage forms, is often the limitation of food intake, drinking, or restriction of the tongue movements at a certain period after administration (of a dosage form). The use of mucoadhesive gels, which are characterized by high viscosity, can slow down the gel penetration of nanoparticles through the gel to a mucosa. Usage

The drug or API will normally leave, exit or be released from the carrier (or system) as a result of mass action, usually after adherence or contact of the carrier with a mucosa. This occurs usually due to the difference in chemical potential, or the high concentration of the drug/ API. This can drive or move the drug or API out of the carrier to, or towards, the mucosa, and suitably across the membrane. Thus, on application of the carrier or system to the mucosa, there will exist a difference in chemical potential or concentration of the API/drug. It is this difference in chemical potential or concentration that will normally result in the drug or API moving out of the carrier and into the mucosa.

Reservoir

The nanoscaffold or matrix suitably comprises nanofibres, having a high or very high surface area.

The nanoscaffold or matrix can therefore act as a reservoir or depot for the drug or API, suitably at a high density or high concentration. Indeed, the nanoscaffold or matrix may be "sponge like" and/or be able to carry a (high) drug load.

Suitably the nanoscaffold will have a high drug or API loading. However, it may also be able to release the drug and/or API relatively quickly. This can be achieved by making the nanoscaffold, or matrix, hyper-porous. The nanoscaffold will therefore contain a high number of pores, gaps or interstitial spaces. This can be formed or achieved by using a preferred preparation process, namely electrospinning. Suitably therefore the nanoscaffold is prepared by electrospinning (to compromise nanofibers), or it comprises at least parts of which (such as nanofibers) that have been electrospun.

Preferably the nanoscaffold comprises (electrospun) nanofibres and/or suitably these are highly cross-linked. This can be achieved if the nanofibres have been electrospun, or prepared by electrospinning. Preferably, the nanofibres are (present) in a random direction and/or are multidirectional. Thus, the nanoscaffold may comprise nanofibres which, if they are electrospun, or formed by electrospinning, may be in a random direction or be in a multitude of directions (see, for example, Figure 10A, B and C). Suitably the nanofibres are prepared by random deposition, or are randomly deposited, and suitably there is little or no fiber aggregation.

Suitably the nanoscaffold (or nanofibres) comprise (or a re made of) natural substances, for example natural polymers, preferably a peptide, polypeptide or chain of amino acids. Preferably such a peptide comprises fibroin, advantageously silk fibroin. Other naturally occurring polymer(s) may be used, such as a carbohydrate, polysaccharide, for example chitosan.

The drug or API is suitably in the form of a particle, such as a nanoparticle. Or, put another way, the nanoscaffold or nanofibres comprise particles (such as nanoparticles) which comprise the API or drug. Suitably there is hydrophilic (as opposed to hydrophobic) or H (hydrogen) bonding or forces between the nanofibres and the nanoparticles. This is a relatively weak polar or electrostatic interaction between (charged and/or polar groups within) the nanofibres and nanoparticles. These relatively weak (polar or electrostatic) interactions mean that the nanoparticles, containing the drug and/or API, can be relatively easily and quickly released from the nanoscaffold. Thus, preferably there are (predominantly) relatively weak interactions, such as weak polar and/or electrostatic interactions, between the nanofibres and the nanoparticles.

Suitably the nanofibres comprise polypeptides which have low complexity crystalline domains and/or Gly-X repeats where X is Ala, Ser and/or Tyr. Suitably the peptides have a predominant or higher proportion of Ser, Ala and/or Gly amino acids. Suitably the polypeptide can form beta sheets, such as beta "rope".

Suitably the nanofiber is relatively hydrophilic and/or has a low water contact angle. This parameter was measured in the Examples is described on page 51. Suitably the water contact angle, for example measured using this method, is less than 100, less than 80, less than 60 degrees, and/or preferably above 10 degrees. Suitably the pore size, namely the interstitial gap, is from 1 to 10 μιη (between the fibres). Suitably the nanoscaffold or matrix comprises a mesh, for example random fibres.

The relative size of the (nano) particles and the (nano) fibres can be important. Suitably the nanoparticles are approximately 1/5 to 1/20 size (in approximate diameter) of the diameter/width or the nanofiber, such as from 1/10 to 1/15. Thus, expressed a different way, the ratio of the diameter (or thickness) of the nanoparticles:nanofibres is from 1 :30, 1 :20 or l : 10 to 1 :2, 1 :4 or 1 :6. Electrospun nanofibres

A further aspect of the present invention relates to the electrospun fibres that are used in, or comprise, the nanoscaffold. The invention in another aspect therefore additionally relates to a naturally occurring polymer that has been electrospun, in other words an electrospun natural fiber or natural polymer. Preferably the polymer is a (poly)peptide or repeating amino acids, or a carbohydrate saccharide or polyamide.

Preferred features and/or characteristics of one aspect of the invention are applicable to another aspect mutatis mutandis.

Brief description of drawings

Fig. 1 illustrates several embodiments of a mucoadhesive carrier in the shape of a round disc according to Example 1.

In the first two embodiments, in section A-A or in section B-B, several possibilities of deposition of layers are shown (1 - nanofibrous layer 2 - mucoadhesive layer, 3 - cover layer 4 - intermediate layer). The third and fourth embodiments (sections C-C and D-D) show the situation when the nanofiber layer is deposited directly on the cover layer and the mucoadhesive layer is also deposited directly on the cover layer in the parts where the nanofibrous layer is not deposited. Fig. 2. Nanofibrous mucoadhesive film (Example 3). A: The scheme shows the bottom view (left) and cross-section (right) of possible variants for construction of nanofibrous mucoadhesive film; B: Photograph of nanofibrous mucoadhesive film, left - design for large animal experiments (pig), and right - design for small animal experiments (mice) (centimetre scale). Nanofibrous reservoir layer (asterisk), mucoadhesive layer (arrow); C: Scanning electron microscopy (SEM) picture showing individual layers of a three-layered nanofibrous mucoadhesive film. The mucoadhesive layer creates a peripheral adhesive ring surrounding the central part on which the electrospun nanofibrous reservoir layer is fixed. Nanofibrous reservoir layer (asterisk), mucoadhesive layer (arrow); D: Cross-section of mucoadhesive layer observed in its native state after freezing (cryo-SEM). Nanofibrous reservoir layer (asterisk), mucoadhesive layer (square) and backing layer (arrow); E: Detail of cross-section of nanofibrous mucoadhesive film, arrow indicates the Eudragit® L 100-55 backing layer (arrow); F: Detail of cross-section of nanofibrous mucoadhesive film, arrow indicates the connection of the mucoadhesive layer and the nanofibrous reservoir layer.

Fig. 3 shows a diagram of the dissolution rate of the cover layer (Example 1).

Eudragit L 100-55 layer as an example of soluble cover layer completely dissolves within 30 minutes, Ethylcelulose layer as an example of non-soluble cover layer. The dissolution process was monitored as increase of fluorescent signal from released water-soluble fluorescent dye (6-Carboxyfluorescein) from the cover layer into dissolution medium.

Fig. 4 Overall view of the mucoadhesive system with a nanofibrous layer for the delivery of nanoparticles (Example 1). A - On the right hand side of the image, an overlapping adhesive margin of the system for nanoparticle delivery can be seen. The nanofibrous layer in the middle serves as a reservoir for nanoparticles. B - Detail of the nanofiber layer deposited on the surface of the mucoadhesive film.

Fig. 5 A and B. Administration of the mucoadhesive carrier of particles to sublingual mucosa in a human (Example 2). The picture was taken 2 hours after the administration; the tongue movements when speaking or ingesting food did not affect the adhesive properties. Fig. 6 Ex -vivo model for evaluation of nanoparticle penetration into porcine oral mucosa (Example 11); A: Schema of ex-vivo penetration test. Surface of the mucosa was continuously wetted during incubation at 37 °C B: Nanofibrous mucoadhesive film on the porcine sublingual mucosa. Rhodamine-labelled liposomes were seen to create depo on the mucosa at the site of film application over all 2-hour period of treatment. The dotted circular lines are illustrative and border the mucoadhesive ring surrounding the central nanofibrous reservoir layer; C: A view of porcine sublingual mucosa on which free nanoparticles were administered. The pink colour was not observable after a short time of the test because of the nanoparticle wash out. Fig. 7 shows liposomes and PLGA-PEG nanoparticles adsorbed to nanofibrous reservoir layer observed by cryo-SEM, SEM, TEM and Confocal Laser Scanning Microscopy

Penetration and adsorption of liposomes and PLGA-PEG nanoparticles into the nanofibrous layer. (Example 4, Example 11). A: Cross-section of an SF nanofibrous mucoadhesive film with liposome-loaded reservoir layer (cryo-SEM). Backing layer (black arrow), mucoadhesive layer (white square) and the nanofibrous reservoir layer (white arrow); B: Detail of SF nanofibrous reservoir layer loaded with liposomes (SEM, deceleration mode). Liposomes (white arrow), nanofiber (asterisk). Note the spherical shape of liposomes adsorbed on nanofibres and the fact that their aqueous internal cavities are intact in spite of being in vacuum for 10 mins or more; C: Detail of liposomes adsorbed to the PCL nanofibrous reservoir layer (TEM). Liposomes (white arrow) nanofiber (asterisk). Note the appearance of liposomes as hollow spheres demonstrating their preserved vesicular structure. The shell-like structures of liposomes with aqueous interiors are clearly visible; D: GFP-proteoliposomes adsorbed to PCL nanofibrous layer (nanofibres were counterstained with fluorescent lipid lissamine rhodamine-PE). Liposomes (white arrow), nanofiber (asterisk); E: SF nanofibrous reservoir layer with adsorbed PLGA-PEG nanoparticles (SEM); F: Detailed picture of PLGA- PEG nanoparticles adsorbed onto the surface of the SF nanofibrous reservoir layer (SEM). PLGA-PEG nanoparticles (black arrowheads) and nanofiber (black asterisk).

Fig. 8 Penetration and adsorption of liposomes with surface-bound model green fluorescent protein (GFP) into the nanofibrous layer prepared from polycaprolactone (PCL). Images of liposomes and nanofibers were taken by confocal microscopy (Example 4). A) Nanofibers labelled using the fluorescent marker lissamine-rhodamine. B) Adsorbed liposomes with surface-bound GFP. C) Overlap of images A and B. D) Detailed view of liposomes with GFP.

Fig. 9 Penetration of the hydrophilic low-molecular weight fluorescent marker 6- carboxyfluorescein and PLGA-PEG labelled nanoparticles into the nanofibrous layer (Example 4). A) Penetration of the fluorescent marker 6-carboxyfluorescein to the nanofibrous layer prepared from a mixture of chitosan/PEO labelled with a fluorescent marker lissamine-rhodamine. B) Penetration of the fluorescent marker 6-carboxyfluorescein into the nanofibrous layer made from PCL. C) Penetration of PLGA-PEG nanoparticles labelled with 3,3'-dioctadecyloxacarbocyanine perchl orate (DiOC18) to the PCL nanofibrous layer.

Fig. 10 Penetration and adsorption of the PLGA-PEG nanoparticles into the nanofibrous layer (Example 4). A) Nanofibrous layer prepared from a mixture of polymers chitosan/PEO. B, C) Adsorbed PLGA-PEG nanoparticles on nanofibers. D) In greater detail. E) PLGA-PEG nanoparticles labelled with the fluorescent label lissamine-rhodamine adsorbed onto nanofibers. F) PLGA-PEG nanoparticles impregnated in the nanofiber layer; particles are placed in the space between nanofibers; they are not adsorbed directly onto the nanofibers.

Fig. 11 Size and zeta-potential of nanoparticles formed by lactic and glycolic acid copolymer, surface-modified by polyethylene glycol (PLGA-PEG) (Example 4). A) nanoparticle size (Z- diameter 135 nm, polydispersity index: 0.144); B) zeta-potential of PLGA-PEG nanoparticles (-2.21 mV).

Fig. 12 The effect of material used, modification of the surface of nanofibres to the amount of the released lissamine-rhodamine PLGA-PEG nanoparticles from nanoscaffold layer (%) (Example 5).

Fig. 13 The effect of material used, modification of the surface of nanofibres to the amount of the released lissamine-rhodamine liposomes from nanofibres (%) (Example 5).

Fig. 14 Adsorption of microparticles of "bacterial ghosts" (BG) type on a nanofibrous layer made from PCL (Example 6). A) Microparticles of "bacterial ghosts" type impregnated in the nanofibrous layer (scanning electron microscopy, SEM). B) Detailed view of a microparticle adsorbed on the surface of a nanofiber (SEM). C) Fluorescently labelled microparticles of "bacterial ghosts" type impregnated in the nanofibrous layer (confocal microscopy). D) Transverse view; impregnation of particles can be observed along the entire nanofibrous layer.

Fig. 15 Cross-section of porcine sublingual mucosa. Penetration of PEG liposomes into the porcine sublingual mucosa can be observed (Example 7). A) Nuclei B) Fluorescently labelled liposomes, C) Overlap of A) and B), Actin is also labelled.

Fig. 16. Tight adhesion of nanofibrous mucoadhesive film to oral mucosa A-G: Adhered film after 2 h of contact incubation with porcine sublingual mucosa as observed by Cryo-SEM. A: the cube-like sample of sublingual tissue with a nanofibrous mucoadhesive film in a cryo- SEM chamber at -130 °C; B: tight adhesion of a nanofibrous reservoir layer to sublingual mucosa; C: detailed picture of tight adhesion; D & E; Illustrative pictures with clearly visible -mucoadhesive layer (upper) and silk fibroin nanofibrous reservoir layer (lower) detached from the mucosal surface during the sample preparation; F: detailed picture of silk fibroin nanofibrous reservoir layer adhered to the mucosal surface. The mesh of mucin fibres is visible in the bottom part. PLGA-PEG (poly(lactic-co-glycolic acid)-polyethylene glycol) nanoparticles are found adsorbed on the surface of nanofibres as well as permeating the mesh of mucin fibres (arrows in the detailed inset); G: general picture of silk fibroin nanofibrous mucoadhesive film and adjacent mucosa after 2 h incubation. A residue of gel mucoadhesive layer is seen above the nanofibrous layer. The gap between the nanofibrous reservoir layer and the mucosa is an artefact generated during sample preparation. A mucin mesh is clearly distinguished on the surface of the mucosa in the middle part of the image. At the bottom of the image, particular epithelial cells are visible.

Fig. 17 Cross-section of porcine buccal mucosa. Penetration of PEG liposomes into porcine buccal mucosa can be observed (Example 7). A) Nuclei B) Fluorescently labelled liposomes, C) Overlap of A) and B), Actin is also labelled.

Fig. 18 A cross-section through porcine sublingual mucosa. Penetration of PLGA-PEG nanoparticles into the porcine sublingual mucosa (formulation containing 1% sodium deoxycholate as accelerator of absorption of nanoparticles) can be seen (Example 7). A) Nuclei, B) fluorescently labelled liposomes, C) Overlap of A) and B), Actin is also labelled.

Fig. 19 Cross-section of porcine sublingual mucosa. The effect of adding 1% sodium deoxycholate on the penetration of PLGA-PEG nanoparticles into sublingual porcine mucosa can be observed (Example 7). A) Penetration of PLGA-PEG nanoparticles from the nanofiber layer to the sublingual mucosa. B) Penetration of PLGA-PEG nanoparticles from the nanofiber layer with the addition of 1% sodium deoxycholate to the sublingual mucosa. Fig. 20 Nanofiber mucoadhesive carrier of particles used for the experiments on mice (Example 8). A) The entire system with a nanofiber layer in the middle and an overlapping adhesive edge. B) Detail of a nanofibrous layer with adsorbed PLGA-PEG nanoparticles.

Fig. 21. Cross-section of murine sublingual mucosa after in vivo administration of PLGA- PEG nanoparticles (Example 8, Example 11). A) Specialized immune cells are present in large quantities in the sublingual area. Cells are labelled with the anti-HLA-DR antibody (yellow colour). B) White arrows indicate the PLGA-PEG particles which have been taken up by phagocytic cells (stained cell nuclei). C) A detailed view confirms the internalization of particles within a phagocytic cell.

Fig. 22 The amount of nanoparticles released from the lyophilized nanofibrous layer (Example 9). The effect of 20% sucrose, 1% deoxycholate and a mixture of sucrose and deoxycholate (final concentration 20% and 1%) present in the solution being deposited, on the number of particles released from the nanofibrous layer after lyophilisation.

Fig. 23. Lymph node delivery of PLGA-PEG nanoparticle applied onto sublingual mucosa via nanofibrous mucoadhesive films (Example 11); A: Cross-section of porcine sublingual mucosa after 2 h incubation in-vivo. PLGA-PEG loaded nanofibrous mucoadhesive film (top layer), PLGA-PEG nanoparticles (dots) penetrating through sublingual tissue, nuclei of epithelial cell (blue); B: Regional lymph node. PLGA-PEG nanoparticles on the cross-section of a regional lymph node (cortex); C: PLGA-PEG nanoparticles on the cross-section of a regional lymph node (cortex), cell nuclei were counterstained by Sytox Blue; D: Regional lymph node. PLGA-PEG particles in the cortex (T-cell area) of a regional lymph node. In the bottom part of the picture, a primary lymph nodule (folliculi lymphatici corticales) is clearly distinguished. Staining of actin and nuclei; E: Regional lymph node. PLGA-PEG particles in the cortex of a regional lymph node. A high number of SLA II-positive B-cells is typical of a primary lymph nodule. pAPC in cortex containing fluorescent nanoparticles; F: Detailed picture of pAPC transporting endocytosed fluorescent PLGA-PEG nanoparticles into T-cell region in a lymph node. pAPC was stained by monoclonal antibody against SLA. Stromal cells in the cortex are stained by antibodies against actin. Fig. 24. Schematic presentation of the principle for improving delivery of drug-delivery and vaccination nanoparticles by means of a nanofibrous mucoadhesive film (Example 11). High adsorption loading capacity of nanofibrous material ensures high concentration of nanoparticles to be reached after the rapid release from reservoir layer to the limited volume of the fluids at the application site. Protecting backing layer prevents removal of nanoparticles from the site of administration by flow of mucosal secretions and saliva. Concentration gradient is formed, then exerts a "pressure" on the mucosal layer so rapidly enabling the formation of a nanoparticle diffusion potential across the mucosal surface into the submucosa. The different fate of nanoparticles (local/systemic delivery) is based on its physicochemical properties and presence of targeting moieties. Dendritic cells (DCs) present in the submucosa are then free to capture vaccination nanoparticles for delivery to the local lymphatic nodes that drain the submucosal zone of application. Vaccination nanoparticles not captured by DCs, are otherwise free to diffuse through the submucosa reaching lymphatic capillaries by means of which they drift to the local lymph nodes for capture by professional antigen- presenting cells.

Fig. 25 Scheme of needless electrospinning setup (Example 11)

Fig. 26 Profilometry of particular layers of nanofibrous mucoadhesive film (Example 11); A: Visualisation of porous structure of reservoir nanofibrous silk fibroin layer; B: Measurement of the step height between the surface of the mucoadhesive layer and the surface of the nanofibrous reservoir layer (silk fibroin) (profile measured in white line segment); C: Measurement of amplitude of wave-like profile of Eudragit® L 100-55 backing layer (profile measured in white line segment).

Fig. 27 Nanofibrous reservoir layers preformulated from different materials by electrospinning (Example 11); A: Polycaprolactone, B: Silk fibroin, C: chitosan-PEO

Fig. 28 Characterisation of model mucus penetrating nanoparticles (Example 11); A: size distribution of PLGA-PEG nanoparticles (DLS); B: size distribution of liposomes (DLS); C: size distribution of PLGA-PEG nanoparticles (NTA); D: size distribution of liposomes (NTA); E: TEM analysis of PLGA-PEG nanoparticles; F: TEM analysis of liposomes; G: inserted Table - characteristics of nanoparticles.

Fig. 29. Determination of the quantity of released nanoparticles from reservoir nanofibrous layer in-vitro/ex-vivo A: Extent of liposome release from different types of nanofibrous reservoir layer (SF is silk fibroin) determined by means of the modified dissolution test; B: Extent of PLGA-PEG nanoparticle release from different types of nanofibrous reservoir layer by modified dissolution test; C: Representative picture of PLGA-PEG loaded onto a silk fibroin nanofibrous reservoir layer as observed by SEM before the dissolution test; D: Representative image of silk fibroin nanofibrous layer after the dissolution test; E: Cross- section of cryo-altered porcine sublingual mucosa with PLGA-PEG nanoparticles penetrated into mucosal tissue after 2 h incubation with a nanofibrous mucoadhesive film; F: Cross- section of cryo-altered sublingual mucosa penetrated with PLGA-PEG nanoparticles with 2% sodium deoxycholate after 2 h Fig. 30. Oral mucosa after 2 h ex-vivo incubation at 37 °C with nanofibrous mucoadhesive films containing rhodamine labelled nanoparticles (Example 11); A: Images demonstrating the paracellular pathway of nanoparticle penetration through porcine sublingual epithelium (PLGA-PEG nanoparticles were used throughout); B. Images showing penetration of PLGA- PEG nanoparticles and liposomes into porcine oral mucosa tissue after release from nanofibrous mucoadhesive films. Fig. 31 Internalisation of fluorescence labelled PLGA-PEG nanoparticles by human dendritic cells (Example 11). Suspension of fluorescently labelled PLGA-PEG nanoparticles (middle picture) were added to human DCs prepared from adhesive peripheral mononuclear cells (monocytes) cultivated in Cell Gro® medium supplemented with IL-4 and GM-CSF (CellGenix, Germany). After 3-hour incubation of DCs with nanoparticles were fixed with 0,5 % paraformaldehyde and stained with FITC-conjugated monoclonal antibodies against ULA- DR cell surface marker (lower picture). The nucleus was counterstained using Sytox Blue dye (upper picture). Internalisation of nanoparticles into cells was clearly observed. Fig. 32 Internalisation of fluorescence labelled liposomes by human dendritic cell (Example 11). Suspension of fluorescently labelled liposomes (picture in the middle) were added to human DCs prepared from adhesive peripheral mononuclear cells (monocytes) cultivated in Cell Gro® medium supplemented with 11-4 and GM-CSF (CellGenix, Germany). After 3-hour incubation of DCs with nanoparticles were fixed with 0,5 % paraformaldehyde and stained with FITC-conjugated monoclonal antibodies against HLA-DR cell surface marker (picture on the right). The nucleus was counterstained using Sytox Blue dye (picture on the left). Internalisation of nanoparticles into cells was clearly observed.

Fig. 33 Modified Franz diffusion cell (Example 12) Donor and acceptor compartment are separated by prepared oral mucosa (sublingual or buccal). The mucoadhesive dosage form is applied onto the mucosa and the fluid is sampled from acceptor as well as donor chamber during the experiment. The amout of released particles in both compartments are determined.

Fig. 34 Characterisation of model PLGA-PEG mucus penetrating nanoparticles (Example 12); A: size distribution of PLGA-PEG nanoparticles (DLS); C: size distribution of PLGA-PEG nanoparticles (NTA); E: TEM analysis of PLGA-PEG nanoparticles; G: inserted Table - characteristics of nanoparticles.

Fig. 35 The efficiency of transmucosal Ps penetration into receptor chamber vs. unwanted release of NPs into the donor chamber using a nanofibrous mucoadhesive film. The figure clearly demonstrates the efficiency in transmucosal transport of NPs using nanofibrous mucoadhesive films. Fig. 36 The efficiency of transmucosal NPs penetration into receptor chamber vs. unwanted release of NPs into the donor chamber using a (standard) double-layered mucoadhesive film. The efficiency is much lower in comparison to nanofibrous mucoadhesive film.

Fig. 37 The efficiency of transmucosal NPs penetration into receptor chamber vs. unwanted release of NPs into the donor chamber using a mucoadhesive gel. The efficiency is much lower in comparison to nanofibrous mucoadhesive film

Fig. 38 Ratio of NPs in donor vs. receptor chamber after 60 and 240 mins (effectivity of unidirectional NP delivery), without any penetration enhancer. The figure clearly demonstrates the highest efficiency in transmucosal transport of NPs using nanofibrous mucoadhesive films as compared to other dosage forms. Note, that the numbers represent the ratio between receptor and donor compartments at time intervals of 60 and 240 mins and do not take in to the account the absolute amount of NP applied to oral mucosa

Fig. 39 The comparison of efficiency of NP transmucosal delivery over time using different dosage forms demonstrating clear variations in the efficiency of NP transmucosal delivery depending on those different dosage forms used. Fig. 40 The effect of NP concentration on the efficiency of NP transmucosal delivery mediated by nanofibrous mucoadhesive films

Fig. 41 The effect of the sodium deoxycholate (10%) as penetration enhancer on the efficiency of NP transmucosal delivery using nanofibrous mucoadhesive films (NP concentration 15 mg/ml)

Fig. 42 Effect of lyophylisation on efficiency of NP transmucosal delivery mediated by nanofibrous mucoadhesive films Fig. 43 Rate of NP transmucosal penetration of NPs using different mucoadhesive dosage forms The following Examples of carrying out the invention are provided purely for illustration and should not be regarded as limiting. Example 1: Preparation of a mucoadhesive nanofibrous carrier of particles

The mucoadhesive nanofibrous carrier for administration of particles to a mucosal surface consists of several layers. The mucoadhesive layer 2 is a layer that provides adhesion of the whole system to the mucosa and consists of a film of different thickness prepared from substances with mucoadhesive properties or their mixtures. Typically, this layer, from the side intended for the orientation into the oral cavity, is covered with a cover layer 3 which is either slowly soluble, or insoluble in the environment of the oral cavity and has no adhesive properties. It is formed by some film-forming substances used in pharmacy. A film-forming agent is deposited to the mucoadhesive layer as a spray containing the polymer solution and appropriate other substances (e.g. softeners). The nanofibrous layer 1 serves as a reservoir of nanoparticles where the nanoparticles are placed in the space among nanofibres and/or on the surface of nanofibres from where they are released into the mucosa. The nanofibrous layer is deposited to the adhesive layer a) by in situ formation, using the electrostatic spinning process, b) by depositing a preformed nanofiber layer on the mucoadhesive layer.

Fig. 1 illustrates several embodiments of the mucoadhesive carrier in the shape of a round disc. In the first two embodiments, in section A-A or in section B-B, several possibilities of deposition of layers are shown (1 - nanofibrous layer 2 - mucoadhesive layer, 3 - cover layer 4 - intermediate layer). The third and fourth embodiments (sections C-C and D-D) show the situation when the nanofiber layer is deposited directly on the cover layer and the mucoadhesive layer is also deposited directly on the cover layer in the parts where the nanofibrous layer is not deposited. More preferred embodiments of the mucoadhesive carrier are shown in Fig 2. Preparation of the mucoadhesive layer: The layer providing adhesion of the whole system to the target oral mucosa was prepared from a mixture of biocompatible mucoadhesive polymers Carbopol 934P (Noveon, Inc., USA) and Methocel K4M (HPMC) (Colorcon, GB). 300 mg of Carbopol 934P and 100 mg of HPMC were dissolved in 25 ml of water. 20 ml of glycerine, serving as a plasticizer, was added to the polymer solution. The method of evaporating the solvent of the polymer solution at 45 °C was used to produce an adhesive film with suitable mechanical properties. The thickness of the obtained film is approximately 85 μπι (see Fig. 2D).

Preparation of the nanofibrous layers: An example is the production of two types of nanofibrous layers: Chitosan/polyethylenoxide (PEO):

The 8% solution of chitosan and the 4% solution of PEO were prepared separately. Chitosan was dissolved in 10% citric acid and PEO was dissolved in distilled water. Both solutions were stirred separately (for 10 hours) using electromagnetic stirrer. In the next operation, sodium chloride at a concentration of 0.85 mol/1 was added to the solution of PEO. Subsequently, the polymer solution of chitosan and solution of PEO were combined in order to obtain a solution where the chitosan/PEO weight ratio might be 8:2. The polymer solution was then electrostatically spun partly to a nonwoven material of the spun-bond type (PEGATEX S 30 g/m², anti-static, blue) and partly to the mucoadhesive layer in such a way to form a nanofiber layer in three different square weights, namely 5, 10 and 15 g/m². In order to obtain different surface weights of the nanofibrous layer, it was necessary to electrostatically spin the polymer solution for varying times. The conditions of electrospinning were: the distance of the earthed collector from the electrode 10 cm, voltage 50 kV, temperature 2FC, humidity 60%. Polycaprolactone (PCL):

Commercially available PCL was dissolved in a mixture of solvents acetone/ethanol (7/3 v/v) at a concentration of 16%. Electrospinning was carried out under the following conditions: the distance of the earthed collector from the electrode 10 cm, voltage 50 kV, temperature 21°C. Electrospinning was carried out using a nonwoven material of the spun-bond type (PEGATEX S 30 g/m², anti-static, blue). The square weight of the resultant nanofibrous layer was 5 g/m² or 15 g/m². Thickness of the polycaprolactone nanofiber layer having the square weight of 15 g/m² is in the range of 55-70 μιη. Thickness of the polycaprolactone nanofiber layer having a square weight of 5 g/m² is in the range of 10-18 μιη.

Depositing of the non-adhesive cover layer: The prepared mucoadhesive layer has been on one side coated with a non-adhesive cover layer. The non-adhesive cover layer has improved mechanical properties, which prevented the adhesion of the nanofiber mucoadhesive carriers to other than the target site during the administration. The cover layer should facilitate the administration of the whole system to the target site and handling with it, extend the interval of the residing time of the system on the mucosa and reduce or completely block the diffusion of nanoparticles from the administration site to the space of the oral cavity.

The cover layer may be formed by a polymer soluble in the oral cavity environment or an insoluble polymer. Mechanical properties and the dissolution rate of the carrier are affected by the choice of the cover layer. Eudragit^® 100-55L was chosen as an example of coating having suitable mechanical properties soluble in the oral cavity environment. Eudragit^® 100-55L was applied by spraying in the form of a 1% ethanol solution with the addition of propylene glycol as a plasticizer (0.25 g Eudragit^® 100-55L, 35 μΐ of propylene glycol, 25 ml ethanol (96%)). The resulting coating thickness depending on the amount of the applied polymer solution was in the order of several hundred nanometers to μπι units (see Fig. 2E).

To prepare a cover layer insoluble in the oral cavity environment, ethyl cellulose polymer was used as an example. The polymer was applied as a spray of a 2.5% solution of ethyl cellulose in ethanol (0.25 g ethyl cellulose, 17.5 μΐ propylene glycol, and 10 ml ethanol (96%)) on the surface of the mucoadhesive layer (Fig. 2). For faster evaporation of the solvent, the mucoadhesive layer was placed on a heated plate at 50°C. Both ethyl cellulose and Eudragit^® 100-55L are commonly used in preparing human pharmaceutical formulations. They are nontoxic and safe. Determination of the dissolution rate of the covering polymeric film: To determine the dissolution rate of the covering polymer film, hydrophilic fluorescent label 6- carboxy fluorescein, used for labelling of the nanofiber mucoadhesive carrier, was added to the polymer solution. The carrier was placed on the bottom of a 100 ml vessel. Phosphate buffer with pH 6.0 was chosen as the dissolution medium. The dissolution rate of the cover layer was determined as the concentration of 6-carboxyfluorescein buffer increasing in time. While the coating prepared from Eudragit 100-55L completely dissolved in approximately 30 minutes, coating prepared from ethyl cellulose remained almost undissolved during the monitoring period (see Fig. 3).

Assembling the nanofiber layer with the mucoadhesive layer: The nanofibrous layer made from a mixture of polymers chitosan/PEO with the thickness of 10 μπι was attached by pressing against the mucoadhesive layer (mixture of HPMC and Carbopol 934P in a weight ratio 1 :3) after slight moistening of the mucoadhesive layer by water steam. Whereas no penetration of the mucoadhesive layer into the layer of nanofibres occurs, their tight and mechanically durable attachment develops. The elasticity of the two layers will ensure intimate contact with the target tissue.

Preparation of the nanofibrous layer by the electrostatic spinning process onto the mucoadhesive layer: The nanofibrous layer can be prepared by the process of electrostatic spinning of a polymer solution directly on the mucoadhesive layer. The mucoadhesive layer was placed on a collector, below which a spinning electrode was located. The polymer solution was dispensed to the spinning electrode at the volume of 1.5 ml and spun directly onto the mucoadhesive layer under the following conditions: the distance of the gathering collector from the earthed electrode was 10 cm, voltage 30 kV. In both examples of the attachment of the layers of the carrier, mechanically durable attachment is achieved, without affecting the structure and function of either layer.

Fig. 4 illustrates a system of the nanofiber layer and the mucoadhesive layer; Fig. 2 shows a cross-section of the nanofiber layer system, nanofiber layer and cover layer.

Example 2: Method of the mucoadhesive carrier administration onto the mucosa A mucoadhesive nanofiber carrier of particles is administered onto the oral mucosa, particularly sublingual and buccal which is not keratinized in humans. The nanofiber mucoadhesive carrier is placed on a finger with the non-adhesive side against the finger and by a slight pressure is applied to the target site in the oral cavity, for example to the underside of the tongue (sublingual mucosa) or to the buccal mucosa, for approximately 5 seconds, before adhesion is created between the mucoadhesive side of the system and the mucosa. Alternatively, a suitable applicator can be used. The applicator is particularly advantageous in veterinary medicine. It was verified that 3 hours after the administration, the tongue movements during speaking or ingesting food did not affect the adhesive properties of the carrier (Fig. 5).

Example 3: Ex-vivo administration of the mucoadhesive carrier on the mucosa

Porcine sublingual mucosa is a model of qualities that are very close to humans. After the removal from a freshly killed animal, sublingual mucosa and buccal mucosa were washed with saline and were used immediately for the administration of nanoparticles using a carrier. Firstly, the nanofiber layer of the carrier was saturated with a solution of liposomes or nanoparticles prepared from the mixture of PLGA and PLGA-PEG polymers with a concentration of 20 mg/ml. Further, the carrier with liposomes or nanoparticles was placed on a finger with the non-adhesive side against the finger and exerting a slight pressure for about 5 seconds it was applied to the target site. In order to study the penetration of liposomes and PLGA nanoparticles into the tissue, the system administered to the mucosa was incubated in a moist chamber at 37°C for 4 hours. Mucosal surface was kept moistened with saline to simulate saliva production. The situation after the 4-hour incubation is shown in Fig. 6.

Preparation of liposomes: Liposomes were prepared by a lipid film hydration method. The final liposome size was achieved by extrusion through polycarbonate filters with pores of a defined size of 100 nm. Composition of liposomes (fluorescently labelled): 10 mol% (1-methoxy -poly ethylene glycol 2000)-N-carboxy-l,2-distearoyl-s«-glycero-3-phosphoethanolamine (PEG²⁰⁰⁰-DSPE); 89.5 mol% egg phosphatidylcholine (EPC); 0.5 mol% l,2-dioleoyl-s«-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rhoda) .

Example 4: Impregnation of the nanofibrous layer with nanoparticles

The nanofibrous layer was impregnated with a suspension of nanoparticles (liposomes or PLGA-PEG nanoparticles). Depending on the properties of nanoparticles, the nanofiber layer and on the method used, the nanoparticles were adsorbed onto the nanofibre surface or formed inclusions in the space between the nanofibers (Figs 7, 8, 9 and 10). The carrier of nanoparticles prepared in this way is applied immediately after the deposition of nanoparticles. It is also possible to stabilize the particles in the nanofibrous layer for long- term storage. The particles are kept in the nanofibrous layer after the solvent evaporation. However, stabilization of particles by the lyophylisation process with cryoprotectants added into the solution of nanoparticles appears more advantageous.

One possibility of applying nanoparticles on the nanofiber layer is the application of nanoparticles in the solution after assembling the nanofiber layer with the adhesive layer. This was carried out by turning down the system with its non-adhesive side after which the solution of nanoparticles was applied onto the surface of the nanofibrous layer. In this way the nanoparticles spontaneously spread evenly and impregnated the nanofibrous layer. For impregnating a nanofiber layer having an area of 0.5 cm² and a thickness of 15 μπι, 2 μΐ of particle suspension was used. The concentration of the nanoparticles (liposomes or PLGA- PEG) was 20 mg/ml. Another application method is the immersion of the nanofiber layer in the solution of nanoparticles. This was performed by immersion of the nanofibrous layer into a solution of nanoparticles of the required concentration. Where required by the nanofiber properties, a tray-shaped ultrasonic bath was used to facilitate the impregnation of particles. For impregnating a nanofiber layer having an area of 0.5 cm² and a thickness of 15 μπι, 100 μΐ of a solution of nanoparticles (liposomes, liposomes with surface-bound model protein or PLGA-PEG nanoparticles) was used at a concentration of 20 mg/ml. The nanofibrous layer was immersed to this solution for 5 minutes. In the case of penetration of PLGA-PEG nanoparticles, the vial containing the solution of nanoparticles was immersed in a tray-shaped ultrasonic bath to facilitate impregnation.

Facilitation of the impregnation of particles into the nanofibrous layer and influencing the rate of adsorption of nanoparticles onto the surface of nanofibers was be achieved by physical or chemical modification of the surface of nanofibers.

Preparation of liposomes: Liposomes were prepared by a lipid film hydration method. The final liposome size was achieved by extrusion through polycarbonate filters with pores of a defined size of 100 nm. For the preparation of liposomes with surface-bound protein, the prepared liposomes were mixed with the recombinant His-tagged protein GFP in a defined ratio. The protein was bound to the surface of liposomes by means of metallochelation.

Composition of PEG liposomes: 10 mol% (1-methoxy-poly ethylene glycol 2000)-N-carboxy- l,2-distearoyl-s«-glycero-3-phosphoethanolamine (PEG²⁰⁰⁰-DSPE); 90 mol% egg phosphatidylcholine (EPC).

Composition of liposomes for surface modification by GFP protein: 5 mol% of 1,2-di- (9Z- octadecenoyl)-s«-glycero-3-[N-(5 -amino- 1-carboxypentyl) imino-diacetic acid succinyl] nickel (II) salt (DOGS-NT A-Ni); 19 mol% of l-hexadecanoyl-2-(9Z-octadecenoyl)-s«- glycero-3-phospho-(l'-rac-glycerol) (POPG); 76 mol% EPC.

Preparation of PLGA-PEG nanoparticles: Nanoparticles were prepared by dissolving 25 mg of PLGA (lactic acid: glycolic acid (50:50), Mw 30,000 to 60,000) (Sigma-Aldrich) and PLGA-PEG (PEG Mw 5,000, PLGA Mw 55,000) (Sigma-Aldrich) in 1 ml of dichloromethane. 1 ml of the organic phase was emulsified in 5 ml of 0.7% sodium cholate by sonication at 70% amplitude, by 1 -second pulses for 5 min. The emulsion obtained in this way was diluted with 20 ml of 0.5% sodium cholate, and the organic phase was removed from the emulsion in an evaporator under reduced pressure. Large aggregated particles were removed by centrifugation at 500 rpm/min. From the resulting nanoparticle suspension, excess cholate was removed by diafiltration (Spectrum). The particles were concentrated to the desired concentration in the same way. The size (A) and zeta potential (B) of PLGA-PEG nanoparticles were measured by the dynamic light scattering method; the result is shown in Fig. 11.

Example 5: Release of nanoparticles from the nanofibrous layer - the effect of the material used and the effect of surface modification of nanofibres on the release of nanoparticles

Interactions of nanoparticles and nanofibers in the carrier matrix is affected by the surface properties of nanoparticles and nanofibers. The release rate and releasable amount of nanoparticles can be affected by the polymer used for the production of nanofibres and its subsequent surface modification. Surface properties of nanoparticles can also be modified in order to improve their release from the nanofibrous layer. Nanoparticles must meet a number of criteria so that they might be able to pass through the mucosal barriers and, therefore, it is very advantageous to modify the surface properties of nanofibers by chemical or physical means. Listed below are examples of possible modifications:

1) Chemical treatment of nanofibers made from PCL

2) Physical adsorption of surfactants onto the surface of nanofibers made from PCL

The numbers of PLGA-PEG nanoparticles and PEG liposomes released from the nanofibrous layer penetrated by the given type of nanoparticles were monitored.

Furthermore, some surfactants (e.g. sodium lauryl sulphate, sodium deoxycholate and others, known as absorption accelerators) increase permeability of the mucosa for drugs and nanoparticles (Fig. 12). Then, two functions can be fulfilled by the present surfactants. They increase the penetration of nanoparticles applied to the mucous membrane by changing the mucosal barrier functions and enhancing the release of nanoparticles from the nanofiber layer.

Chemical modification of nanofibers: The nanofibers made from PCL are of hydrophobic character. To increase wettability and limit the hydrophobic interactions with nanoparticles, their surface was modified by immersion of the nanofibrous layer in 3 M NaOH for 10 min. The nanofibrous layer was then rinsed with water several times (Fig. 13). Deposition of the nanoparticles solution of on the nanofiber layer: The nanofibrous layer of PCL was penetrated by its immersion in a solution of PLGA-PEG nanoparticles or PEG liposomes at a concentration of 20 mg/ml for 5 min.

Releasing of nanoparticles from the nanofiber layer to the solution: The nanofibrous PCL layer of a round shape with the surface area of 0.5 cm² penetrated by the solution of PLGA- PEG nanoparticles was immediately placed in 0.5 ml of aqueous solution. The nanofibrous layer was incubated for 30 minutes under gentle shaking. The solution obtained was diluted as necessary to match the parameters for nanoparticle concentration measurement using the chosen method.

Determination of the number of the released nanoparticles: The number and size of released PEG-PLGA nanoparticles were determined by "Nanoparticle tracking analysis" (NanoSight, Malvern, UK). The amount of released liposomes was determined as the solution fluorescence intensity after incubation of the nanofibrous layer measured at excitation of 560 nm and emission of 583 nm. The obtained data was adjusted according to the dilution factor of the measured solution and the amount of the released particles was calculated (%). Adsorption of surfactants: The effect of adsorption of surfactants onto the surface of nanofibers was studied after penetration of the nanoparticle penetration of the nanofibrous layer. The solution of nanoparticles contained sodium deoxycholate at a concentration of 1%. In a second embodiment, the nanofibrous layer was first immersed in a solution of 1% sodium deoxycholate, rinsed several times with water and dried. Then it was impregnated with a solution of nanoparticles.

Example 6: Adsorption of microparticles of the "bacterial ghosts" type

Microparticles are also be used as vaccine delivery systems. One type of such microparticles are empty bacterial envelopes termed as "bacterial ghosts" (BG).

BG are non-pathogenic particles derived from bacterial cells. They contain the bacterial cell wall, including antigenic structures, against which a specific immune response is induced. The intracellular content is removed for example by osmotic shock, and therefore the particles obtained in this way are unable to further propagate. Due to the natural presence of a number of substances recognized by the immune system known as danger signals, such particles provide a complex signal for inducing a specific immune response against the antigenic structures present. Bacterial ghosts can potentially be used as vaccine particles for mucosal administration.

Fluorescent labelling of "bacterial ghosts": Bacterial ghosts (prepared from Escherichia coli) were ultrasonically dispersed in water. Fluorescent marker DiOC18 dissolved in ethanol was added to the suspension of bacterial particles, the mixture was further ultrasonicated for 1 min so that the fluorescent marker might incorporate in the wall of the particles. Centrifugation and washing removed the excess fluorescent marker.

Fluorescent labelling of nanofibers: The nanofibrous layer prepared from PCL was labelled with the fluorescent dye lissamine-rhodamine. The nanofibrous layer was penetrated with labelled bacterial particles. Adsorption of bacterial particles on the nanofibrous layer was confirmed by the techniques of scanning electron microscopy and confocal microscopy (Fig. 14). Preparation of the nanofibrous layer with microparticles of the "bacterial ghosts" type: The suspension of "bacterial ghosts" was prepared from 1 mg of BG lyophilisate in 1 ml of water using the tray-shaped ultrasound. The nanofibrous layer was immersed in this suspension and the vial was placed into the tray-shaped ultrasound for 5 minutes. Example 7: Penetration of nanoparticles (PLGA or liposomes) into the sublingual and buccal porcine mucosa after their release from the nanofiber mucoadhesive particle carrier

Penetration of nanoparticles from a nanofiber mucoadhesive carrier to the mucosa was confirmed in cross sections after incubation of the carrier adhered to freshly excised porcine sublingual and buccal mucosa (Figs 15, 16 and 17). Preparation of the nanoparticle carrier : The nanoparticle carrier was prepared according to the procedure described in Example 1. The nanofibrous layer was impregnated with a solution of PLGA-PEG nanoparticles or PEG liposomes labelled with lissamine-rhodamine. To facilitate the penetration of nanoparticles into the mucosa, a suspension of PLGA-PEG nanoparticles in 1% sodium deoxycholate was used for impregnation of the nanofiber layer (Figs 18, 19).

Application of nanoparticles by means of a mucoadhesive system: A nanofiber mucoadhesive carrier with fluorescently labelled nanoparticles (PLGA-PEG or PEG liposomes dyed with lissamine-rhodamine) was administered to freshly excised sublingual mucosa by gentle pressure (see Fig. 6). Tissue samples were incubated at 37°C for 4 hours. Then they were then quickly frozen in liquid nitrogen and stored at -75°C.

Preparation of tissue cross-sections: Cross-sections of 10-μπι thickness were cut on a Cryo- cut instrument (Leica), fixed with acetone, and if needed, nuclei (blue, Sytox Blue) and actin (green, Alexa Fluor^® 488 Phalloidin) were stained.

Example 8: Penetration of PLGA-PEG nanoparticles into mouse sublingual mucosa

The sublingual mucosa contains different types of immune cells involved in immune response of the body and in inducing tolerance to the present antigens. Many types of particles (nanoparticles/microparticles) are suitable carriers of antigens. The particles allow us to combine antigens with immunomodulatory agents capable of influencing the resulting immune response. The structure of sublingual mucosa differs between rodents, humans and pigs. It differs mainly in the degree of keratinization, which is a barrier to penetration of nanoparticles into the mucosa.

In an in vivo mouse model, no spontaneous penetration of PLGA nanoparticles into the sublingual mucosa was observed in contrast to porcine mucosa (see Figs. 20 and 21). In in- vivo experiments, physiological functions of the immune system cells are not suppressed as it is in ex vivo experiments carried out on porcine mucous membranes (see Figs. 15 to 19) and it is possible to observe particle the internalisation by phagocytic cells involved in the regulation of the immune response/tolerance.

In the experiment, the occurrence of large amounts of MHC Il-positive cells capable of phagocytosis was confirmed in the mouse sublingual area where the mucoadhesive system for nanoparticles was administered (Fig. 21A). Phagocytosis of PLGA-PEG nanoparticles by specialized cells was also confirmed.

Preparation of PLGA-PEG nanoparticles: see Example, Embodiment 3

Administration of nanoparticles by means of the carrier into the mouse sublingual area: Adhesive system with a 4-mm diameter was slightly pressed against the mucosa in the sublingual area of a mouse. Administration time was 4 hours. After this time, the mouse was sacrificed and frozen in n-heptane of a temperature of -70°C.

Preparation of a cross-section of tissue: see Example, Embodiment 7

Evaluation of the experiment: Internalization of nanoparticles in specialized cells was localized by confocal microscopy.

Example 9: Lyophilisation of the nanofibrous layer with impregnated nanoparticles

If required by the nature of the nanoparticles and/or physiologically active substances carried by them, long-term stability of nanoparticles and/or the carried physiologically active substances which penetrated into the nanofibrous layer, can be achieved by lyophilisation or by simple drying.

Depending on the nature of the nanoparticles, the amount of particles releasable from the nanofibrous layer can be considerably influenced by the addition of other substances to the solution of nanoparticles. The addition of cryopreservation agents (for example saccharides, such as sucrose, trehalose) and/or surfactants appear advantageous (Fig. 22).

Preparation of a PLGA-PEG nanoparticle suspension: PLGA-PEG nanoparticles (nanoparticle preparation see Example, Embodiment 3) were prepared as a suspension in water, 1% sodium deoxycholate, 20% sucrose or a mixture of 1% sodium deoxycholate and 20%) sucrose.

Preparation of the nanofibrous layer: The amount of particles releasable from the matrix after lyophilisation was monitored in nanofibrous layers prepared from PCL.

Penetration of PLGA-PEG nanoparticles into the nanofiber matrix: Nanofiber matrix made from polycaprolactone, with the layer thickness of 15 μπι and an area of 0.5cm², was penetrated by PLGA-PEG nanoparticles by immersion into the prepared solution and sonication in an ultrasonic bath for 5 minutes.

Lyophilization of the nanofibrous layer with PLGA-PEG nanoparticles: After penetration, the samples were immediately frozen on dry ice so as to prevent drying of the solution. Frozen samples were lyophilized. The effect of cryoprotectants and surfactants was tested for the amount of releasable nanoparticles from the nanofibrous layer.

Release of PEG-PLGA nanoparticles from the nanofiber layer: Individual lyophilised nanofiber layers with nanoparticles were transferred into 500 μΐ of MilliQ -filtered water (20nm Anotop filter, Millipore). The release of nanoparticles was carried out for 30 minutes while stirring on a shaker.

Determination of the number of released nanoparticles: Released amount and size of the PLGA-PEG nanoparticles was determined by "Nanoparticle Tracking Analysis" (NanoSight, Malvern, UK). Example 10: Penetration of nanoparticles (PLGA and liposomes) into the sublingual mucosa of a piglet in vivo after their administration, using a nanofiber mucoadhesive carrier

Preparation of the nanoparticle carrier: The nanoparticle carrier was prepared as described in Example, Embodiment 1. The nanofibrous layer was impregnated with a solution of PLGA- PEG nanoparticles or PEG liposomes labelled by lissamine-rhodamine. To facilitate penetration of nanoparticles into the mucosa, PLGA-PEG nanoparticle suspension in 1% sodium deoxycholate was used for impregnation of the nanofiber layer.

Administration of nanoparticles by the mucoadhesive system: A nanofiber mucoadhesive carrier with fluorescently labelled nanoparticles (PLGA-PEG rhodamine or PEG liposomes) was administered to the sublingual mucosa or buccal mucosa of a piglet (15 kg) applying a slight finger pressure. During the administration, the piglet was under general anaesthesia (injection of a short-acting anaesthetic). After two hours, the pig was again put into general anaesthesia and euthanized. Adjacent tissue with the particle carrier and a regional lymph node were excised and cross sections of tissues were prepared for the evaluation.

In-vivo penetration of particles (Fig. 23): Penetration of nanoparticles from the nanofiber mucoadhesive carrier into the mucosa (Figure 23 A) and regional lymph nodes (Figure 23B-E) was confirmed in cross-sections after oral mucosal administration of the carrier to the sublingual or buccal mucosa of the pig. The internalisation of PLGA-PEG nanoparticles by porcine antigen presenting cells was also confirmed (Figure 23F).

Example 11: Nanofibre-based mucoadhesive films were prepared for oromucosal administration of nanocarriers used for delivery of drugs and vaccines

The mucoadhesive film consisted of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and alike plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allow for high levels of nanoparticle loading.

The nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers can promote prolonged release of nanoparticles into submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. The system appears appropriate for use with oral mucosa, especially for sublingual and buccal tissues. Trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. Effective mucosal delivery of nanoparticles has become an important area with respect to development of safe and effective mucosal vaccines. As the number of new macromolecular drug and vaccine candidates (peptides, proteins, nucleic acids, antigens, allergens, virus like particles, etc.) formulated into nanoparticle delivery systems increase, there is the unmet need for its effective delivery. Sublingual and buccal region remains important site of administration of macromolecules, especially due to its mild environment, relatively high permeability and non-invasive and safe application route.

Although formulations of mucus penetrating nanoparticles capable of free diffusion through mucus barrier have been suggested, little is known about the mucosal and transmucosal delivery of nanoparticles (covering mucus diffusion barrier as well as epithelial absorption barriers). Biological testing of nanoparticle-based drugs and vaccines delivery systems is limited to application in the form of free suspension, or formulated into muco-adhesive gels without using any nanoparticle-delivery device ensuring prolonged formation of depo on the site of application. Application of such formulations often requires the sedation of experimental animals throughout the time of experimentation.

In this Example, we make a mucoadhesive platform based on nanofibrous reservoir for nanoparticles and macromolecules. This platform takes advantage of biocompatible nanofibre-based materials (large surface area and internal volume, flexibility, stability and adhesiveness) to ensure controllable and prolonged mucosal delivery of vaccines and drugs owing to formation of the depot at mucosal surface and tunable release of nanoparticles from the nanofibre-based matrices (see Fig. 24). Unique properties of nanofibrous materials distinguishes the platform from standard mucoadhesive films. This platform may be especially advantageous for antigen delivery in mucosal immunisation and immunotherapy. The platform can be used for development of other technologies (e.g. printed vaccine technologies) appropriate for the production of cheap, self-administrable, non-invasive prophylactic and anti-allergic vaccines. Nanofibre-based mucoadhesive films were prepared for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. An electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and alike plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allow for high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. The system appears appropriate for use with oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model.

The oromucosal route of administration is an alternative route for drug-delivery and vaccine delivery. This has been used successfully for local and systemic delivery of low-molecular- weight substances with a rapid onset of pharmacological effects, and also for drugs formulated into controlled-release systems (i.e. Onsolis®). So too, sublingual immunotherapy that makes use of allergens and antigens for the treatment of allergies and sublingual vaccination strategies, have all been explored. In addition, mucosal, especially sublingual delivery of macromolecular drug and antigen-delivery nanoparticles has become an important topic of recent research [1]. In general, high permeability, a lack of enzymatic barriers, mild pH values, easy access for self-administration, and opportunities to bypass first-pass metabolism, all make the non-keratinized oral regions attractive sites for the administration of drugs and/or vaccines [2]. Oral mucosa and most especially the sublingual region are densely populated with specialized dendritic cells and the adjacent submucosal tissue is drained with lymphatic vessels bringing absorbed antigens as well as migrating dendritic cells into regional lymph nodes. Thus, oral cavity and especially sublingual region is a potentially favourable site for inducing a specific immune response or tolerance towards given antigens and allergens. The sublingual mucosa in particular has been recognized in particular as a suitable immuno-inductive area giving the opportunity for safe and efficient mucosal vaccination or immunomodulation [3].

However, although mucosal surfaces are the main route of pathogen entry, the induction of effective mucosal immunity is still a challenge for researchers in the field of vaccinology [4]. In order to induce an effective immune response not only is the vaccine system itself important but so too is the method of application [5]. Factors that limit successful administration and delivery of nano-based therapeutic systems include the mucus layer itself on the surface of oral mucosa, continuous saliva production, and epithelial absorption barriers [6]. Typically, administered drugs/antigens are efficiently removed by mucus clearance mechanisms and systemic absorption, precluding a prolonged local drug presence [7]. New approaches and formulations that utilize muco-adhesives, mucus penetrating particles, or absorption enhancers can enable effective transmucosal delivery of macromolecular therapeutics [8] and nanoparticle-based delivery systems [6]. In addition, a variety of dosage forms have been developed to face these obstacles for oral mucosal delivery - including muco-adhesive tablets, muco-adhesive oral films, fast dissolving films as well as liquid formulations and sprays. Many nanoparticle-based delivery systems including polymeric and lipid-based nanoparticles have also been tested as mucosal drug-delivery and vaccination nanoparticle systems, especially for protein, peptide, and nucleic acid delivery [9].

Successful delivery of drug-delivery or vaccination nanoparticles through mucosal tissues represents a multi disciplinary problem that embraces aspects of mucosal physiology and mucosal barrier properties, nanotechnology and nanoparticle surface chemistries. So the problem falls to pharmaceutical sciences with particular reference to appropriate pharmaceutical formulations, dose regimens, drug-delivery devices, and proper elucidation of therapeutic approach [10]. Here we describe for the first time the combination of electrospun nanofibrous reservoir layers prepared by electrospinning, with muco-adhesive layers that together appear to represent an important new dosage platform for effective administration of drug-delivery and vaccination nanoparticles into the sublingual and buccal mucosa. Electrospinning is a simple and efficient technique to produce nanofibers [11]. It utilizes a high electrostatic field to generate nanofibers from a fluid. Electrospun nanofibers often show large surface-to-weight (volume) ratio, high porosity, and excellent pore interconnectivity [12]. These unique features allow electrospun nanofibers have extensive applications in diverse areas including filtration, wound healing, cosmetic, drug delivery systems, and medicine [13, 14]

The exemplified nanofibrous muco-adhesive films can avoid fast clearance of nanoparticles from sites of application, can maintain a long-term concentration gradient of nanoparticles at the mucosal surface, and may ensure unidirectional diffusion of nanoparticles towards mucosal surfaces by means of an impermeable surface layer(s) that faces the oral cavity.

Preparation of nanofibrous mucoadhesive films

Each three-layered film prepared consisted of 1) a muco-adhesive layer, 2) a backing layer and 3) an electrospun nanofibrous reservoir layer. In the first step, the upper surface of a prepared muco-adhesive layer was coated with polymer to form a backing layer. In the second step the bottom side of each muco-adhesive layer was wetted with a vapour stream and a corresponding nanofibrous layer immediately pressed against this muco-adhesive layer. Mucoadhesive layers were prepared as follows: initially Carbopol 934P (Lubrizol Advanced Materials, Cleveland, USA) and hydroxypropyl methylcelulose K4M (HPMC) (Colorcon Limited, UK) were combined in a 2: 1 (w/w) ratio in water giving a viscous opaque solution that was supplemented with glycerol 15 % (w/w). The combination mixture was then treated by sonicated to remove air bubbles and required volumes were poured out into plastic Petri dishes. Excess water was removed by evaporation at room temperature for 48 h leading to the formation of the desired mucoadhesive layers.

Non-adhesive backing layers were formed by the spraying of a 2% ethanolic solution of Eudragit® L 100-55 (soluble) directly onto the surface of a given muco-adhesive layer. During the spraying, each Petri dish with a given mucoadhesive layer was heated to 50 °C to accelerate the evaporation of solvent.

Nanofibrous reservoir layer All nanofibrous reservoir layers were prepared using a roller electrospinning device (see Fig. 25). The device contains a rotating cylinder, 145 mm in length and 20 mm in diameter, partially immersed in a blended polymer solution reservoir attached to a positive electrode. Blended polymer solutions were electrospun at a high voltage of 50 kV with the cylinder rotating at ~ 15 rpm in order to become rapidly coated with polymer solution followed by the electrospinning process itself and the creation of new materials. By this method each electrospun nanofibrous reservoir layer was collected on backing material (PEGATEX S 30 g/m²) that was moving along a negative collector electrode at a velocity of 30 mm/min. Electrospinning was carried out at a distance of 100 mm; air temperature 21 °C, and air humidity 60 ± 2 %.

Silk fibroin nanofibrous reservoir layer

Raw silk cocoons were degummed twice with 0.1 M of sodium carbonate and 0.5% of standard reference detergent at 100 °C for 30 min, rinsed with warm water to remove the sericin from the surface of the fibre and then dried at room temperature. Silk fibroin solution (SF) was prepared by dissolving the degummed silk fibres in formic acid (98%). The formic acid solution used in the process contains 3 wt% calcium chloride. The silk fibroin concentration was fixed at 12 wt%. The SF solution was magnetically stirred at room temperature overnight, then electrospun at conditions described above.

The silk fibroin nanofibrous reservoir layers were immersed in ethanol for 30 min to induce crystallization of the silk fibroin and reduce the water solubility of the nanofibrous reservoir layers. After drying at room temperature, the treated nanofibrous reservoir layers were immersed in distilled water overnight, which was followed by rinsing in distilled water to remove residual salts. Afterwards, the nanofibrous reservoir layer were air-dried.

Chitosan-PEO nanofibrous reservoir layer

Chitosan (viscosity 10 cP, 5 wt% in 1% acetic acid and a degree of deacetylation of 0.8) was purchased from Wako Pure Chemical Industries, polyethylene-oxide (PEO, average Mw ~ 400.000 g/mol by gel permeation chromatography) was obtained from Sigma-Aldrich and used as received. Deionized water with sodium chloride was used to prepare PEO polymer solution. Sodium chloride was added to an aqueous solution of 4 wt% PEO at 0.24 mol/L. Chitosan was then dissolved in 10 wt% citric acid to achieve a polymer concentration of 8 wt%. Chitosan and PEO solutions were afterwards blended and stirred at room temperature overnight at a volume ratio of 8/2 (chitosan/PEO). This blended chitosan/PEO polymer solution was electrospun at conditions described above.

Polycaprolactone nanofibrous reservoir layer

Polycaprolactone (Mw 80 000 g/mol, Sigma Aldrich) solution was prepared by dissolving PCL pellets in the solvent mixture acetone/ethanol (7/3 v/v) by means of overnight stirring at room temperature. The total polymer concentration was fixed at 16 wt%. Thereafter the blended PCL polymer solution was electrospun at conditions described above.

Preparation of nanoparticles

Liposomes

Liposomes were prepared by the lipid film hydration method. The composition of liposomes was 10 mol% (1-methoxy-polyethylene glycol 2000)-N-carboxy-l,2-distearoyl-s«-glycero-3- phosphoethanolamine (PEG²⁰⁰⁰-DSPE); 89.5 % egg phosphatidylcholine (EPC) (Avantilipids), 0.5 mol% l,2-dioleoyl-5«-glycero-3-phosphoethanolamine-/V-(lissamine rhodamine B sulfonyl) (DOPE-Rhoda) (Avantilipids) . After hydration by PBS, liposomes were extruded using a 200 nm polycarbonate filter (Millipore, USA).

Metallochelating nanoliposomes were prepared by the lipid film hydration method and were extruded using a 200 nm polycarbonate filter. The composition of liposomes was 5 mol% 1,2- dioleoyl-5«-glycero-3-[/V-(5-amino-l-carboxypentyl)imino-diacetic acid succinyl] nickel (II) salt (DOGS-NT A-Ni) (Avantilipids), 71mol% EPC, 19mol% l-palmitoyl-2-oleoyl-sw- glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (POPG) (Avantilipids). Prepared liposomes were mixed with His-tagged GFP protein (20ug GFP per lmg of lipid). The preparation is described in detail in Masek et al [30].

PLGA-PEG nanoparticles

PLGA particles were prepared by the emulsifying method. Briefly, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide), Mw 55.000Da, PEG average 5.000Da, poly(D,L-lactide-co-glycolide) (lactide:glycolide (5:5), mol wt 30.000-60.000) (Sigma) and l,2-dioleoyl-5«-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE- Rhoda) (Avantilipids) were dissolved in 1ml chloroform and emulsified in 5 ml of 1% sodium deoxycholate solution using the ultrasound device SONOPULS HD 3100 (Bandelin, Germany). The particles were formed by evaporation from chloroform using a rotary evaporator. The excess of sodium deoxycholate was removed and PLGA particles were concentrated in one step using MikroKros hollow fibre modules (50 kDa) (Spectrum, USA).

Profilometry and macroscopic morphology analysis of nanofibrous mucoadhesive film

Scanning electron microscope (Hitachi8010, Hitachi, Japan) was used to investigate the macroscopic morphology of nanofibrous mucoadhesive films, the surface texture of nanofibrous layers as well as nanoparticles adsorbed to the surfaces of fibres. A cross-section of a nanofibrous mucoadhesive film was prepared at -130 °C. The sample was coated with Pt- Pd and observed by cryo-SEM at -130 °C. Adsorption of fluorescent-labelled nanoparticles to nanofibres was observed by confocal microscopy (Leica SP2, Germany) and the adsorption of liposomes was observed by TEM (Philips Morgagni). Detailed measurements of the thicknesses of nanofibrous reservoir layers, texture analyses of these layers and of backing layers were performed using an optical surface metrology system (Leica DCM8, Germany) under confocal mode (5 OX objective). Analysis of the diameter of nanofibrous reservoir layers

Nanofibrous reservoir layers were observed under a scanning electron microscope (SEM) Vega 3 (Tescan, Czech Republic) at an accelerated voltage of 20 kV. Prior to measurements, all the samples were sputter-coated (Q150R ES, Quorum Technologies Ltd., England) with gold at a thickness of 7 nm. The SEM images were analyzed with NIS-Elements AR software. The average fibre diameter and its distribution were determined from 150 random fibres.

Measurement of water contact angle

The hydrophilicity of the nanofibrous reservoir layers was evaluated by water contact angle measurements using a See System E instrument (Advex Instruments, s. r. o., Czech Republic). A distilled water droplet of the size of 10 μΕ was placed carefully onto the surface of the nanofibres at room temperature. After a period of 20 s, the contact angle was recorded. The mean value and standard deviation (SD) were also calculated through testing at ten different positions on the same sample. Determination of in-vitro release of nanoparticles from nanofibrous reservoir layer

PLGA-PEG or liposomal nanoparticles (20 mg/ml) were allowed to infiltrate nanofibrous reservoir layers (surface area of 1 cm2) that were dipped into appropriate suspensions for 2 mins. Immediately after adsorption of particles, nanofibrous reservoir layers were cleansed of excess fluids and placed into PBS buffer (1 ml) for 30 mins. The final PBS solution of PLGA nanoparticles was diluted and the concentrations of released nanoparticles were calculated using a NanoSight 500 instrument (Malvern Instruments, UK). As an alternative method, the final PBS solution of fluorescence-labelled nanoparticles was diluted and the ratios of bound to released nanoparticles were calculated from differential fluorescence intensities measured at the emission wavelength of 560 nm using a spectrofluorimeter LS55B (Perkin Elmer).

Determination of ex-vivo release of nanoparticles from nanofibrous reservoir layer

The ability of the electrospun nanofibrous reservoir layer to release associated nanoparticles in real conditions (moistened mucosal tissue surfaces with limited water volumes, typical of viscous mucus layers etc.) was confirmed by 2 h incubation of nanofibrous mucoadhesive films with cryo-altered mucosa having reduced barrier functions at 37° C45. The release of nanoparticles from nanofibrous reservoir layers was demonstrated by observation of fluorescence signals on cross-sections of adjacent tissues by confocal microscopy. The surface of each mucosa was moistened (flow rate 0.1 ml/min) with PBS during experiments using a linear pump and tubing.

Ex-vivo study of penetration of nanoparticles into tissues

Ex-vivo penetration of nanoparticles into adjacent mucosa was tested on freshly excised porcine sublingual or buccal tissues. Nanofibrous mucoadhesive films with fluorescently labelled nanoparticles were applied to mucosa and incubated for 2 h at 37 °C. The surface of each mucosa was wetted as described above. In-vivo study of penetration of nanoparticles to adjacent mucosa and lymph node delivery

Piglets (15 kg) were anesthetized with the short-term anaesthetic Zoletil (Virbac, France). Multi-layered mucoadhesive film loaded with fluorescently labelled PLGA-PEG nanoparticles in 2% sodium deoxycholate was applied to the porcine sublingual mucosa. After 2 h of incubation, animals were anesthetised and sacrificed by an i. v. -application of T61. Sublingual mucosa samples with nanofibrous mucoadhesive films attached and regional draining lymph nodes were excised, frozen and stored at -80 °C. The tissues were sectioned using Leica Cryocut 1800 Cryostat (Leica, Germany). All sections were stained and observed by confocal microscope (Leica SP2). Nuclei were stained with SytoxBlue (Molecular Probes), Actin was stained with Alexa Fluor® 633 Phalloidin, phagocytic cells were stained with SLA Class II DR Antibody | 2E9/13 (Serotec) and goat anti -mouse secondary Alexa Fluor® 488 conjugate (Abeam). Nanoparticles were stained with lissamine-rhodamine phosphatidylethanolamine (Avanti Polar Lipids, USA).

Cryo-SEM of adhesion of nanofibrous layer onto mucosal surface

2 h post application of mucoadhesive films to piglets, cube-shaped samples of mucosal tissue with adhered mucoadhesive films were removed and cross-sectioned with Cryocut (Cryocut 1800, Leica, Austria) at -20 °C. Subsequently, the samples were transferred under nitrogen atmosphere into the scanning electron microscope (Hitachi SU8010, Hitachi Ltd., Japan). The tightness of adherence of the mucoadhesive layer to mucosa and the structure of nanofibrous mucoadhesive films were observed under cryo-mode by SEM at -130 °C.

Cryo-alteration of porcine mucosa

Freshly excised porcine mucosa was rinsed twice with PBS buffer at room temperature. Subsequently, the rinsed sublingual mucosa samples were freezed-thawed in two cycles to -20 °C. The sublingual mucosa samples were stored at -20 °C until use. Cryo-altered mucosa was used as a positive control to confirm the in-situ release of nanoparticles from nanofibres. Physicochemical analyses of nanoparticles

Nanoparticle sizes and zeta potentials were measured by dynamic light scattering (ZetaSizer Nano ZS, Malvern, UK) at the wavelength of 633 nm. The sizes of nanoparticles and the rate of release of PLGA-PEG nanoparticles were measured using a Nanoparticle Tracking Analysis (NTA) (Nanosight 500, Malvern, UK). The samples were diluted appropriately prior to the measurement. During the NTA measurements, the camera level was set to 7. Captured videos were analysed with a detection threshold set to the value of 10. The morphology of nanoparticles was characterised using TEM. Specimens for TEM analysis were prepared by drop-casting particles on carbon coated copper grids stained with phosphomolybdenic acid solution (2%) and dried at room temperature before observation. Bright field imaging was performed using TEM (Phillips 208 S, FEI, Czech Republic) operating at 80 kV. The experiments on animals were approved by the Ethic committee at the Veterinary Research Institute, Brno and by the Ethic Committee at the Ministry of Agriculture, Czech Republic.

Nanofibrous mucoadhesive film

The main task for any mucoadhesive system used for mucosal delivery of drug-delivery or vaccination nanoparticles is maintaining effective nanoparticle concentrations at the given site of application for a sufficient time span to enable functional nanoparticle-mediated delivery to take place. Hence there is the need to create multi-layered nanofibrous mucoadhesive films for the controlled release and delivery of nanoparticles/macromolecules into the oral mucosa in a unidirectional fashion while avoiding nanoparticle losses from the site of the application due to wash-out by saliva (Fig. 24). This objective was achieved by designing nanofibrous mucoadhesive films with three different layers (see Fig. 2): a nanofibrous reservoir layer (1), a mucoadhesive layer (2), and a backing layer (3). In an alternative embodiment an interlayer (4) was added in between layers 2 and 3.

The nanofibrous layer can serve as reservoir for nanoparticles attached to the surface of nanofibres or embedded into the pores between nanofibres. The mucoadhesive layer was introduced to keep the whole platform affixed at the site of its application for a prolonged time span, adjusted by degree of adhesiveness of the material used for the preparation of the mucoadhesive film layer. The backing layer can prevent diffusion of nanoparticles out of the site of application and to protect both the mucoadhesive layer and the nanofibrous reservoir layer from the effect of saliva and flow of mucosal fluid. Moreover, the backing layer can facilitate the self-administration of the film to the oral mucosa by protecting the surface from sticky properties of the mucoadhesive film itself. The mucoadhesive film layer was prepared by a casting/solvent evaporation technique as described elsewhere [15] adapted for deposition of a backing layer onto the surface of a preformed mucoadhesive layer by spray-drying. We selected the oro-dissolving backing layer composed of Eudragit® L 100-55 (Eudragit® L 100-55 dissolves at pH 5.5 or higher). The oro-dissolving backing layer was introduced for better patient compliance since nanofibrous mucoadhesive films can dissolve or undergo erosion with time without the need for removal. By tuning the oro-dissolving properties of the backing layer, the time of adhesion of a given nanofibrous mucoadhesive film to the oral mucosa can be adjusted. Typically controlled thickness of several hundred nanometres can be achieved (Fig. 2D). In contrast, using a common solvent-casting method, the backing layer of several microns in depth is usually obtained [16-18]. The selected mucoadhesive layer was prepared with a specific composition (Carbopol 934P/HPMC K4M ratio 2/1 w/w) for low swellability and a strong mucoadhesive strength thereby ensuring sufficient mucoadhesive properties [19]. The thicknesses of the prepared mucoadhesive layers were observed to be approx. 80 μπι, as measured by SEM in the cryo-mode (Fig. 2D). Nanofibrous reservoir layers were then affixed onto these backing layer coated, pre-formed mucoadhesive layers. As observed by SEM in the cryo-mode, the structure of a given mucoadhesive layer remained essentially unchanged after coating with a backing layer by spray drying (see the arrow in Fig. 2E), and also after attachment of a nanofibrous reservoir layer by means of a sticking procedure (see the arrow in Fig. 2F). Profilometry was used for visualization of features of particular layers of prepared nanofibrous mucoadhesive film (see Fig. 26).

Electrospun nanofibrous reservoir layer

The internal features of electrospun nanofibrous reservoir layers are useful for adsorption of nanoparticles and macromolecules due to their extremely high surface area and porosity, enabling a high nanoparticle to mass ratio in comparison with reservoir layers constructed from other materials [20]. For example, Jung et al. prepared chitosan nanoparticle/polycaprone composite for sustained drug delivery intended as wound dressing material [21]. In general, well known flexibility of thin electrospun nanofibrous reservoir layers can also be important for intimate contact to be established between nanofibrous mucoadhesive films and mucosal surfaces. Otherwise, adjusting the physical properties of electrospun nanofibrous reservoir layers can be important in order to achieve efficient adsorption of drug-delivery and/or vaccination nanoparticles, and their rapid/controlled release when required. Hydrophobic and ionic interactions are the dominant forces that influence binding and controlled release. Hence, we prepared nanofibrous reservoir layers with three different materials, polycaprolactone (PCL), chitosan-polyethylenoxide (PEO) and silk fibroin (Table 1, Fig. 27).

Weight/m² (g) Nanofibre diameter

Material Contact angle (°)

(nm)

PCL 5 118.77 ± 3.62 114.13 ± 17.87

Silk fibroin 5 43.56 ± 5.07 670.22 ± 123.73

Chitosan-PEO 5 11.69 ± 3.18 169.3 ± 43.3

PCL pre-treated NaOH 5 54.04 ± 5.61 N/A*

PCL pre-treated sodium

5 15.1 ± 2.92 N/A*

deoxycholate

Table 1: Physical properties of nanofibres

In addition, various treatment procedures were employed to change the surface properties of nanofibers. For instance, mild sodium hydroxide or deoxycholate surfactant pre-treatment of PCL nanofibrous reservoir layers were both found to increase layer hydrophilicity (i.e. reduce the contact angle) that might otherwise be relatively hydrophobic in character (Table 1).

Application of nanofibrous mucoadhesive film to human mucosa

The comfort, tolerability, compliance, and durability of adhesion of nanofibrous mucoadhesive films were tested on volunteers. The authors proved for themselves the ability of the nanofibrous mucoadhesive films (without any nanoparticle load) to adhere to a site of application on human oral mucosa for extended time spans. These human subjects were allowed to speak and drink during this period without any restriction. The nanofibrous mucoadhesive films used were able to adhere at least for 2 h in the sublingual region (Fig. 5 A, B). Neither adverse reactions such as local irritation nor uncomfortable feelings were observed or reported in the course of testing including during the period post-treatment period.

Tight adhesion of nanofibrous reservoir layer to oral mucosa

The nanofibrous mucoadhesive films were also shown to adhere tightly onto porcine sublingual mucosa (Fig. 16 A-G). The tight adherence of a nanofibrous mucoadhesive film to the porcine oral sublingual mucosa after 2 h incubation was proved by cryo-SEM on cube- shaped cross-sectioned samples. The benefits of such visible tight adhesion are that drug- delivery and/or vaccination nanoparticles located within the nanofibrous reservoir layer of nanofibrous mucoadhesive films are maintained in close contact to the surface of mucosa and avoid being subject to mucosal self-cleaning effects caused by a continuous flow of saliva.

Mucus penetration by drug-delivery or vaccination nanoparticles

Polymeric nanoparticles that possess a dense low-molecular weight polyethylene glycol (PEG) coating and are known as mucus penetrating particles (MPPs) with an enhanced ability to diffuse through mucus layers [22]. MPPs penetrate through mucus by a mechanism of passive diffusion driven by concentration gradients. The dense, hydrophilic PEG coating prevents interactions of hydrophobic nanoparticle core with mucin mesh and therefore minimises mucoadhesive interactions [23-25]. The inclusion of a PEG layer at the liposome surface also ensures mucus-penetrating properties [26]. PLGA-PEG nanoparticles and PEG containing liposomes were prepared for testing in this study. The morphology of both nanoparticles was studied using TEM. Both types of particles exhibited a regular spherical shape and were prepared essentially monodisperse. The electron micrographs of model PLGA-PEG nanoparticles and liposomes are presented. The observed sizes of both nanoparticle types were in a good agreement with DLS (Dynamic Light Scattering) data as well as Nanoparticle Tracking Analysis (NTA) data. Both liposomes and PLGA-PEG nanoparticles also demonstrated similar size distributions and zeta-potentials (electroneutral) (see Fig. 28). Loading the nanofibrous reservoir layer with nanoparticles

Silk fibroin nanofibrous reservoir layers were loaded with nanoparticle dispersions (liposomal or PLGA-PEG nanoparticles). Nanoparticle dispersion was applied directly to the surface of a given nanofibrous layer and were absorbed immediately and uniformly into the whole volumes of given nanofibrous reservoir layers due to the capillary action. The process of loading affected neither the physical nor the morphological properties of adjoining mucoadhesive film layer (Fig. 7A). Strikingly, we observed a dense and homogenous covering of nanofibre surfaces by nanoparticles (PLGA-PEG or liposomes) as demonstrated by electron microscopy and confocal microscopy (Fig. 7B - F). Furthermore, using GFP- proteoliposomes (Green Fluorescent Protein), a model for proteoliposomal vaccination nanoparticles [27-29], we were unable to observe any signs of nanoparticle aggregation, nor apparent disruption of GFP metallochelation binding to liposome surfaces [30], thereby suggesting that nanofibre adsorption is neither physically or chemically disruptive for lipid- based nanoparticle systems (Fig. 7D).

The release of nanoparticles from nanofibrous reservoir layer

The ability of nanofibrous reservoir layer to release adsorbed nanoparticles from nanofibrous reservoir layers into the surrounding milieu was demonstrated using different types of nanofibres. The tested nanofibrous layers were prepared with all three main biocompatible materials of interest, namely with PCL, chitosan-PEO, and silk fibroin polymers. In addition, PCL nanofibrous reservoir layers were treated with 3M sodium hydroxide for 5 mins, or with sodium deoxycholate to improve the wettability of nanofibre surfaces. These procedures significantly improved wettability as reflected by a decrease of the contact angle (see Table 1). The extent of nanoparticle release from a given type of nanofibrous reservoir layer was determined by calculation of nanoparticle concentrations (measured by the NTA technique) after incubation of nanoparticle-loaded nanofibrous reservoir layers with PBS buffer (Fig. 29A, B). Alternatively, in-vitro release was determined by monitoring changes in fluorescence intensity of lissamine rhodamine-labelled nanoparticles (PLGA or liposomes) after the incubation of nanoparticle-loaded nanofibrous layer in PBS buffer (see Fig. 29). The NTA assay was found to be in good agreement with the fluorescence release assay data for both types of nanoparticles. Moreover, cholate salts are widely used as penetration enhancers (see next paragraph); also sodium deoxycholate can perform dual role.

The extent of nanoparticle release was observed to be nanofibre dependent. Essentially quantitative release of nanoparticles was observed with both silk fibroin and chitosan-PEO nanofibers after 30 mins. On the other hand, when standard PCL nanofibers were used, then significantly lower levels of nanoparticle release were observed (approx. 50 %). Thereafter, when PCL nanofibers were pre-treated with 3M sodium hydroxide solution or with 2% (w/v) sodium deoxycholate, then levels of PLGA-PEG nanoparticle release were also near quantitative. These data are in a good accordance with the reduction of a contact angle value caused by both pre-treatment procedures used (Table 1). The almost complete release of PLGA-PEG nanoparticles from Silk fibroin nanofibers in-vitro was confirmed by observation of nanoparticle-loaded nanofibers before and after the dissolution test using SEM (Fig. 30C, D). In general, nanofibres with hydrophilic surfaces (chitosan-PEO, silk fibroin and PCL treated with NaOH) were all competent to release almost all adsorbed nanoparticles into the surrounding medium. By contrast, non-treated PCL nanofibers with rather more hydrophobic surface properties were not able to effect the quantitative release of PLGA-PEG nanoparticles or liposomes within the given incubation time (see Fig. 29A, B).

However, the conditions occurring during in vitro release test differ significantly from those occurring after the application of the nanofibrous film onto the mucosal surface in vivo. Therefore, the established cryo-altered mucosa test is used as an ideal model giving much less false negative results with regard to the assessment of the potential of mucosal delivery system to release the nanoparticles directly onto the site of application. Cryo-altered mucosa has limited barrier functions while maintaining its general physical properties and anatomical structure [31]. This model was used to confirm the data from in vitro release experiments and for testing the release of PLGA-PEG nanoparticles from electrospun nanofibrous reservoir (Silk fibroin) adhered on cryo-altered mucosa ex vivo. The ex -vivo release test using cryo- altered porcine oral mucosa confirmed the ability of released nanoparticles to reach the mucosal surface and penetrate into deep mucosal tissue (Fig. 29E, F).

The penetration of fluorescently labeled PLGA-PEG nanoparticles into the epithelium of cryo-altered mucosa was investigated. Whereas the fluorescence signal comes predominantly from the upper part of the epithelial tissue in case of PLGA-PEG particles without sodium deoxycholate, incubation of cryo-altered mucosa with sodium deoxycholate makes the fluorescence signal from nanoparticles more uniform (see Fig. 29E, F). This observation is in good accordance with the fact, that barrier of extracellular lipids is located in the upper third of epithelium [32]. It is important note that the mechanism of action of cholate salts lies in solubilisation of the extracellular lipids. So, this type of pharmaceutical excipient acts only if its local concentration is maintained above the critical micellar concentration [33]. This implies that the dosage form should release the nanoparticles as well as excipients in unidirectional manner, it should be in the intimate contact with oral mucosa for prolonged period of time, and it should prevent dilution of the substances by saliva.

For subsequent ex vivo and in vivo experiments, our preference was to use silk fibroin nanofibrous mucoadhesive films.

Mucus penetration of nanoparticles and interactions with mucosal tissue— ex vivo and in vivo models

Dramatic anatomical differences are observed in buccal and sublingual mucosa among species. In general, large animals possess a non-keratinized stratified buccal mucosa, which is more similar to the anatomy of the human mucosa. In terms of availability, thickness, and permeation properties, the porcine buccal mucosa appears to be the most suitable animal model due to the highest similarity to human mucosa [34]. By contrast, rodents possess keratinised mucosa in sublingual region. In spite of this fact, many studies evaluating biological effects in vivo, including sublingual vaccination nanoparticle formulations, are performed on more accessible small animal models (mice, rat) having poorly permeable, keratinised oral mucosa [35]. We used both pig and mouse models with our nanofibrous mucoadhesive films applied to buccal and sublingual mucosa. In our case too nanofibrous mucoadhesive films were designed to have properties appropriate for application to oral region of mice, piglets, and man.

Due to availability and simplicity, the evaluation of the effectiveness of transmucosal delivery of nanoparticles is often tested on ex-vivo tissues. As discussed above, crucial consideration for the permeation test must be adequate tissue storage and isolation before the experiment³¹. Freshly excised mucosal tissues were used for all ex-vivo penetration tests. The penetration tests were started approximately 30 mins after excision and were conducted under physiological conditions in terms of temperature and with continuous moistening of the surface as described in the section Materials and Methods. Ex-vivo study of penetration of nanoparticles to porcine oral mucosa

Ex-vivo penetration of nanoparticles to oral mucosa was tested on freshly excised porcine sublingual and buccal tissues (see Fig. 6). A given nanofibrous mucoadhesive film with pre- loaded nanoparticles was applied to excised mucosa and incubated for 2 h at 37 °C. During the whole experiment, the surface of the mucosa was moistened with PBS buffer to simulate conditions in the oral mucosa. For control purposes, free nanoparticles were applied to excised mucosa. The difference between both routes of application was clearly distinguishable by means of the intensity of pink colouration at the site of application. Whereas nanofibrous mucoadhesive films maintained their fluorescent nanoparticles in the middle of a mucoadhesive ring during the incubation period, free nanoparticles were wash out from mucosal surface after a short time of the test and did not penetrate into mucosa at all (Fig. 6). The penetration of nanoparticles into oral mucosa was confirmed by cross-sectioning of adjacent mucosa and observation with confocal microscopy (see Fig. 30). Freshly excised sublingual and buccal oral mucosa with model PLGA-PEG nanoparticles and PEG²⁰⁰⁰-DSPE liposomes were tested. The penetration to the epithelium of the oral mucosa was observed in all tested samples. The intensity of fluorescence reflects the concentration gradient of nanoparticles diffusing deeper into submucosal tissue. The transport of model nanoparticles in epithelium undergoes the paracellular pathway as clearly demonstrated by confocal microscopy at tissue slices. Fig. 30A shows the paracellular transport of nanoparticles in detail. However, cleaning of mucosal surface by continuous production of saliva, presence of viscous mucus layer and the existence of epithelial barriers are main factors limiting the extrapolating of the results from in vitro dissolution test and ex-vivo tests to real in vivo conditions. Therefore, in vivo study of penetration of nanoparticles into adjacent mucosa was carried out using mouse and porcine animal models.

In-vivo study of penetration of nanoparticles into adjacent mucosa in mouse model In the case of the mouse model, administration of nanoparticles by means of a given nanofibrous mucoadhesive film resulted only rarely in specific cell-associated delivery even if a high concentration of nanoparticles was maintained on the surface of sublingual mucosa for a 2 h period without washing out by saliva (Fig. 2 IB, C). This is in good agreement with the fact, that rodent sublingual mucosa is highly keratinised and thus the permeability for nanoparticles is low. The presence of high concentration of specific immune cells (determined as MHC II positive cells) in this area is clearly visible in Fig. 21A. Accordingly, the porcine model is more appropriate for sublingual delivery experiments given the lower levels of keratinisation and the greater anatomical similarity of porcine sublingual to the human equivalent mucosa [34]. Cells responsible for the transport of nanoparticles and antigens across mouse sublingual mucosa, have been defined by Nagai and co-workers [36].

Mucosal and transmucosal transport of nanoparticles to regional lymph nodes in piglets Owing to strong keratinisation of sublingual mucosa in mouse, the pig model is more appropriate for sublingual delivery experiments because of similarity with the anatomy of human sublingual mucosa [34]. This system is especially suitable for sublingual immunotherapy and vaccine applications. Thus, finally the piglet model was used to evaluate the delivery of PLGA-PEG nanoparticles into lymph nodes. Sodium deoxycholate was tested in this experiment as a penetration enhancer. Endocytosis of fluorescent Ps by antigen presenting cells was demonstrated by histochemical staining of tissue sections and their observation by confocal microscopy.

Cross-sectioned slices from histological samples excised 2 h after the application of nanoparticles demonstrated penetration of nanoparticles into mucosa (Fig. 23A) as well as into regional lymph node (Fig. 23B, C). Some portion was recognised and endocytosed by Swine Leucocyte Antigens (SLA) type II positive cells (porcine antigen presenting cells pAPC) and detected in draining regional lymph nodes (Fig. 23D, E). Also pAPC cells with fluorescent nanoparticles were found in both B and T-cell area in lymph nodes (Fig. 23D, E). This observation is well confirmed by a detail picture of pAPC with endocytosed fluorescent nanoparticles (Fig. 23F). The interaction of human DCs with PLGA-PEG or liposome nanopaticles was studied in vitro to demonstrate their ability to endocytose them and to see their subcellular distribution. Accumulation of fluorescent nanoparticles in the cytoplasm of human DCs was found to be similar to that in porcine DCs (see Fig. 31 and Fig. 32).

In general, successful delivery by the oromucosal route of administration is opposed by mucus and mucosal secretions [37] (diffusion barrier). So too the mucosal epithelium represents another main barriers for transport of nanoparticles and macromolecules into the submucosa [2,37] (absorption barrier). Several strategies were introduced for prolonged or enhanced oromucosal delivery of pharmaceutical substances: mucoadhesive formulations, mucus penetrating particle formulations, fast dissolving films and co-application of penetration enhancers.

PLGA-PEG nanoparticles are the most frequently used polymer nanoparticle options used for transmucosal delivery. PLGA-PEG nanoparticles possess a dense low-molecular weight polyethylene glycol coating and are known as mucus penetrating particles (MPPs) with an enhanced ability to diffuse through mucus layers. MPPs penetrate through mucus by a mechanism of passive diffusion driven by concentration gradients. The dense, hydrophilic PEG coating prevents interactions of the more hydrophobic nanoparticle core with mucin mesh and therefore minimises mucoadhesive interactions [23-25, 38]. Nevertheless, MMPs have been widely used for mucosal delivery of a broad range of macromolecules, for example peptides, proteins, and nucleic acids [26]. The presence of PEG also appears to enhance the lymph node delivery of nanoparticles [23]. Such nanoparticles appear useful as vaccination nanoparticles since they can promote the transport of an encapsulated molecular adjuvant and antigen to the draining lymph nodes. Nanoparticle-mediated accumulation of molecular adjuvants to lymph nodes should enable a significant decrease in dose of molecular adjuvants. This approach has been reported with a number of toll-like receptor (TLR) agonists, including monophosphoryl lipid A (MPLA), CpG DNA, poly (I:C), and small-molecule TLR7/8 compounds [39]. However, while PEGylation may promote tissue penetration of nanoparticles, functional delivery of molecular adjuvants and antigens to APCs can be impaired suggesting the need for nanoparticle triggerability [40] and/or targetability. In general, ideal nanoparticle for mucosal delivery of vaccines/immunotherapeutics may comprise paradigm ABCD nanoparticle structure as described by A.D. Miller: antigen/immunomodulator/API payload of nanoparticle (A- component) surrounded by association lipidic/polymeric material (B-component) forming the AB-core. AB-core requires shielding coatings protecting from interactions of nanoparticle with mucus (C-component), and finally targeting layer (D-components— specific ligands e.g. manan, anti-DEC 205, TLR agonists, etc.) might be added to target/activate specific immune dendritic cells [41].

Above mentioned paradigm is perfectly suited for liposomes, the first nanoparticles which received FDA (Food and Drug Administration) approval for human application and have appeared as marketed products, also represent versatile biocompatible systems composed of biological lipids are. Factors such as the size, the surface charge, and surface modification, lamellarity, presence of targeting moieties etc. govern the penetration of liposomes through various tissues, their bio-distribution their fate in an organism. Liposomes are unique with their ability to encapsulate drugs and antigens of various physical -chemical properties (e.g., molecular weight, hydrophobicity, charge, and shape). The inclusion of a PEG layer at the liposome surface also ensures mucus-penetrating properties [26].

Fortunately, when a given nanofibrous muco-adhesive film and nanoparticles with mucus penetrating properties are used for oro-mucosal delivery, the mucus barrier is much less relevant. On the other hand, the barrier represented by the mucosal epithelium remains a problem. Indeed, effective delivery of nanoparticles across the epithelium is a great challenge, too, and in fact the transfer of nanoparticles across the epithelium requires paracellular pathways involving the penetration through extracellular matrix. This process limits the velocity of diffusion across the epithelium.

A frequent strategy to overcome the epithelium barrier is co-administration of nanoparticles with absorption enhancers [33, 42]. Some of the most commonly used enhancers are cholate salts [34. Accordingly, we tested the mucosal penetration of PLGA-PEG nanoparticles after loading into our nanofibrous mucoadhesive films containing a mixture of 2% deoxycholate sodium. Subsequent data demonstrated unidirectional penetration of nanoparticles into porcine submucosa in vivo and their internalisation into immune cells (see Fig. 21, 23, 30), all interlock to support the view that our nanofibrous mucoadhesive films were appropriately positioned on mucosal surfaces to be excellent reservoirs for the storage and then unidirectional controlled release of drug-delivery or vaccination nanoparticles across a given muscosal epithelial cell barrier. Moreover, due to unidirectional delivery, nanofibrous mucoadhesive platform is supposed to provide taste masking for carried vaccine and drug formulations.

Nanofibrous mucoadhesive films seem to be an ideal dosage form for nanoparticle-based vaccine delivery systems. As reviewed by Dukhin and Labib, the pathway of antigens and nanoparticles to a lymph node can be divided into particular steps. The transport across mucus layer (1), across epithelium and its barriers (2), transport through the interstitial fluids of submucosa (3), and transport through lymphatic capillaries to a lymph node (4). As the first three steps are driven by passive diffusion, the transport through lymphatic ways is driven by so called convective diffusion. This explains why nanoparticles were observed in the lymph nodes after only a few hours post mucosal administration [32].

With respect to vaccines, the endocytosis of nanoparticles by antigen presenting cells and their active translocation to draining lymph nodes also represents another important mechanism involved in the penetration of nanoparticles through mucosal tissue. Accordingly, this high adsorption loading capacity ensures high concentration of nanoparticles to be reached after the rapid release from reservoir layer to the limited volume of the fluids at the application site. This concentration gradient then exerts a "pressure" on the mucosal layer so rapidly enabling the formation of a nanoparticle diffusion potential across the mucosal surface into the submucosa (Fig. 6, 23 A, 24). Dendritic cells present in the submucosa are then free to capture nanoparticles for delivery to the local lymphatic nodes that drain the submucosal zone of application [44]. Nanoparticles not captured by DCs, are otherwise free to diffuse through the submucosa reaching lymphatic capillaries by means of which they drift to the local lymph nodes for capture by professional antigen-presenting cells (pAPCs) [32].

The surface adhesive properties of the nanofibres in our nanofibrous reservoir layers can be modified by virtue of the very materials used to prepare nanofibrous reservoir layers or by down-stream treatment [20, 21] (Fig. 29). Such variations of surface properties, either inherent or chemically induced, ensure that the nanoparticle binding and release characteristics of nanofibrous reservoir layers can in principle be tailor-made or adapted according to the requirements of the corresponding nanoparticle delivery system being used for high concentration binding and chemical potential driven release into the submucosa.

Taking the nanofibrous mucoadhesive film as a whole, if a nanofibrous reservoir layer is central to nanoparticle binding and release, then the properties of the other components should also be optimal. Thus the swelling properties of the neighbouring mucoadhesive film layer should have no direct effect on the binding and release of nanoparticles directed to the mucosal surface. In contrast to our own observations, others have reported that nanoparticles can be incorporated into mucoadhesive film layer and experience a delay in release through the mucosa caused by gradual swelling of the mucoadhesive film and diffusion-limitations [18]. Otherwise, the mucoadhesive film layer needs to be prepared to resist movements of the tongue and the extensive production of saliva by sublingual glands. Furthermore, motional stress is more pronounced in the sublingual region as compared to the buccal mucosal region. Thus, nanofibrous mucoadhesive films should be particularly flexible and thin with sufficient mucoadhesive properties to maintain adherence. The protective backing layer can be modified in the mean of solubility according to specific demands without any influence on the release rate of nanoparticles. The shape, composition, surface area and thickness of mucoadhesive layers all need to be optimized to achieve the desired mucoadhesive properties and time span of adhesion. The mechanical and physical-chemical properties of nanofibrous materials pmay redetermine their application for development and production of mucosal drug delivery systems. Especially nanoparticle drug delivery systems and vaccines may have great potential with respect to non-invasive mucosal applications. Such systems can be superior to gel materials with respect to compatibility with broad range of material intended for incorporation, technology of production and storage conditions.

Summary

Overcoming the barriers to successful carriage of drug-delivery and vaccination nanoparticles into submucosal tissues and draining lymphatic nodes is important for many new drug and vaccination opportunities. The Examples demonstrate on an ex vivo and in vivo pig model how electrospun nanofibrous mucoadhesive films can serve as protective nanoparticle reservoirs for the controlled and sustained delivery of nanoparticles into submucosal tissue and draining lymphatic node sites. Furthermore, the electrospun nanofibrous mucoadhesive films can in principle be variously prepared from different polymeric materials and surface functionalised for use with many different types of nanoparticles. Liposomes, viruses and virus like particles, polymeric and lipid-based nanoparticles, biopolymers (e.g. protein and peptide antigens, plasmid DNA, polysaccharides), molecular adjuvants and pharmaceutical excipients (e.g. enhancers of penetration, mucolytics and cryoprotectants) can be combined with nanofibrous materials to develop an appropriate product for non-invasive mucosal application.

Owing to the potential versatility of these systems, future applications in the development of non-invasive sublingual vaccines and immunotherapeutics may enable the use of therapeutic drug-delivery nanoparticles in the oromucosal environment. The development of industrial- scale production of our nanofibrous mucoadhesive films is now in progress.

Example 12: Biophysical studies of mucosal penetration by nanoparticles as a function of time concentration and The aim here was to determine the effectiveness of penetration of nanoparticles (NPs) through model porcine sublingual mucosa using a nanofibrous mucoadhesive films. The effectiveness of NP penetration using nanofibrous mucoadhesive films was compared to other standard formulations such as a double-layered mucoadhesive film or a mucoadhesive gel (with NPs embedded directly in mucoadhesive layer). The effect of applied NP concentration and the effect of the co-application of a penetration enhancer (sodium deoxycholate) were determined.

Experimental The NPs tested were so called mucus penetrating nanoparticles, based on PLGA/PLGA-PEG polymers. Finally, lyophilised NP-containing formulations was compared to freshly prepared NP-containing nanofibrous mucoadhesive films.

A modified diffusion cell was used for testing (see Fig. 33). The construction was intended to enable the placement of different mucoadhesive formulations (films, gels) on fixed mucosa and to simultaneously determine NP penetration levels into a receptor chamber by passage through freshly excised porcine sublingual mucosa. The amount of NP released into the surrounding milieu of the donor chamber was also measured (as a model for release of NPs into the oral cavity without mucosal penetration). The volume of receptor chamber as well as donor chamber was 2 ml. During experiments, 200 μΐ aliquots from donor and receptor chambers were removed for analysis at regular sampling intervals. The volume of 2 ml was maintained by adding fresh buffer. Results of NPs concentration changes with time were corrected for dilution of NPs during sampling. PLGA-PEG NPs were prepared by emulsification method and were fluorescently labelled (DioC18 or l,2-dioleoyl-s«-glycero-3- phosphoethanolamine-N-(carboxyfluorescein). Characterisation of fluorescent PLGA-PEG NPs is shown (Fig. 34).

Mucoadhesive films were prepared by standard solvent casting method. Mucoadhesive gel was prepared by simple solubilisation of mucoadhesive polymer in buffer containing NPs. The nanofibrous mucoadhesive film used comprised silk fibroin (nanofibrous reservoir layer), carbopol 934P /hydroxypropylmethylcelulose (FIPMC) (2/1, w/w) (mucoadhesive layer), and ethylcellulose (backing layer). For comparison a double layered mucoadhesive film was prepared with carbopol 934P /hydroxypropylmethylcelulose (FIPMC) (2/1, w/w) (mucoadhesive layer; into which NPs were formulated) and ethylcellulose (backing layer). Also for comparison, a mucoadhesive gel of 2% gel of FIPMC was prepared into which NPs were formulated.

Freshly excised porcine sublingual mucosa were used for all experiments (experiments started about 30 - 60 mins after the excision); mucoadhesive gel: NP-containing mucoadhesive gel was applied using pipette tip in the volume of 20 μΐ. The area of application was comparable to the area of nanofibrous reservoir layer used in the film; the double layered mucoadhesive film was carefully pushed on the oral mucosa, the concentration of NP per unit of area was comparable to nanofibrous mucoadhesive film; 5 μΐ of NP solution was applied to the reservoir nanofibrous layer of nanofibrous mucoadhesive film and the film was carefully pushed on the oral mucosa.

Results and Discussion The efficiency of transmucosal Ps penetration into receptor chamber (or compartment) vs. unwanted release into the donor chamber (or compartment) is very impressive using the nanofibrous mucoadhesive film with the vast bulk of NPs penetrating into the receptor chamber with time (Fig. 35). Whereas with both (standard) double layered mucoadhesive film (Fig. 36) and mucoadhesive gel (Fig. 37) unwanted release into the donor chamber was seen predominately with time. This effect is summarized at two time points of 60 and 240 mins (Fig. 38), clearly demonstrating the vastly superior characteristics of the nanofibrous mucoadhesive film for unidirectional NP delivery across an oral mucosal surface, even without the assistance of a chemical penetration enhancer. A complete set of data documenting NP penetration into a receptor chamber (or compartment) as a function of time is also presented using either nanofibrous mucoadhesive film, (standard) double layered mucoadhesive film, or mucoadhesive gel, further emphasising the superiority of the nanofibrous system (Fig. 39).

Experiments with the nanofibrous mucoadhesive film were then performed to show how higher NP loading concentrations (60 mg/ml) also promoted transmucosal NP penetration into the receptor chamber (Fig. 40), as did the presnce of deoxycholate penetration enhancer (Fig. 41). The potential benefits of lyophilisation (in the presence of 20% saccharose) on the performance of nanofibrous mucoadhesive film assisted transmucosal NP penetration was further analysed (Fig. 42). Finally data was collected regarding the rate of transmucosal NPs penetration into separate receptor chambers depending on whether either the nanofibrous mucoadhesive film, the (standard) double layered mucoadhesive film, or the mucoadhesive gel were involved in promoting transmucosal penetration (Fig. 43).

In summary:

1) The ideal ratio between unidirectional transmucosal delivery of NPs and unvanted release of NPs into donor compartment was achieved using nanofibrous mucoadhesive films. 2) Using nanofibrous mucoadhesive films, unidirectional transmucosal penetration of mucus penetrating PLGA-PEG NPs was more effective than using the standard double-layered mucoadhesive film or mucoadhesive gel.

3) Transmucosal delivery of NPs could be concentration dependent (the capacity of sublingual mucosa for NP penetration is not a limiting factor in the range of NP concentrations 15-60mg/ml). Concentrations of NPs used in this experiment showed the dependency as an example, 15-60 mg/ml is not limiting.

4) Transmucosal delivery of NPs was enhanced using sodium deoxycholate (as one example of penetration enhancer)

5) Transmucosal delivery of NP using lyophilised formulations of nanofibrous mucoadhesive films was feasible, but less effective using 20% saccharose solution as compared to freshly prepared NP comprising nanofibrous mucoadhesive films.

6) In terms of the rate of transmucosal penetration of NPs, the nanofibrous mucoadhesive film mediated the fastest NP penetration immediately after application to the mucosal surface - illustrative of the fact that the nanofibrous reservoir layer acts can as a fast release, high concentration depo of NPs for unidirectional release at the point of tight contact with the oral mucosa.

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Claims

1. A mucoadhesive (e.g. particle) carrier, which comprises:

- a nanoscaffold (or matrix) carrying or comprising at least one substance or API (e.g. comprised in the form of particles); and

- a mucoadhesive (layer),

wherein the mucoadhesive (layer), on at least a part of its surface, can adhere (to a mucosa) or overlap with the nanoscaffold.

2. The mucoadhesive carrier according to claim 1, characterized in that:

a) the nanoscaffold contains or has pores having the size of from 10 nm to 1,000 μιη and/or is a nanofibrous layer of a thickness in the range 0.1 to 5,000 μιη; and/or

b) comprises a layer of biocompatible polymers or a mixture thereof.

3. The mucoadhesive carrier according to claim 1 or 2, characterized in that:

a) the mucoadhesive layer (at least partially) overlaps the nanoscaffold, an edge of the mucoadhesive layer overlaps an edge of the nanoscaffold and/or the mucoadhesive layer surrounds the nanoscaffold along an edge; or

b) it is adapted for application onto a target mucosa, optionally the nanoscaffold faces the mucosa (e.g. in the same direction as the mucoadhesive) and/or part of the mucoadhesive layer overlapping the nanoscaffold is adapted to adhesively fix (adhere) the mucoadhesive carrier to the target mucosa.

4. The mucoadhesive carrier according to any one of the preceding claims, characterized in that

a) it further comprises a cover layer (suitably not having mucoadhesive properties) which does not allow permeation of the substance;

b) optionally wherein the order of the layers is either nanoscaffold - mucoadhesive layer - cover layer;

c) the nanoscaffold is adjacent to the cover layer (over at least part of its surface); and/or d) the mucoadhesive layer is adjacent to the cover layer over at least part of its surface.

5. The mucoadhesive carrier according to any one of the preceding claims, characterized in that it further comprises an intermediate layer (preferably not having mucoadhesive properties) which does not allow permeation of the substance therethrough,

preferably said intermediate layer being deposited or located between the nanoscaffold and the mucoadhesive and/or cover layer (to which it may be attached).

6. The mucoadhesive carrier according to claim 4 or 5, characterized in that the cover layer and/or intermediate layer are insoluble (in saliva) and/or dissolution/erosion is prolonged.

7. The mucoadhesive carrier according to any preceding claim, characterized in that the particles:

(a) comprise one or more substance(s)/APIs and/or are in the form of, or comprise a nanoparticle, preferably liposome, solid lipid nanoparticle, polymeric nanoparticle, hybrid polymer-lipid nanopartile, virus like particle or virus, virus pseudotype, dendrimer, microparticle (e.g. pollen, bacteria, bacterial fragments and bacterial ghost, polymeric microparticles) or a macromolecule (e.g. enzyme, toxin, peptide, antigen, allergen extract, antibody or a complex with an antigen); and/or

(b) are suitably anchored to, adsorbed on, embedded or located in the nanoscaffold (mesh); and

(c) are mucosa penetrating.

8. The mucoadhesive carrier according to claim 7, additionally comprising at least component that is (at least one) excipient, preferably an absorption accelerator and/or an excipient that may facilitate release of the particles (carried to the mucosal surface) and/or an excipient that may facilitate penetration of the particles through a mucus layer and/or an excipient that may facilitate penetration of the particles into (deeper) layers of the mucosa, mucosal tissue, draining lymph nodes and/or systemic circulation.

9. The mucoadhesive carrier according to any preceding claim, characterized in that the nanoscaffold comprises nanofibres, whose surface is preferably modified by physical or chemical treatment, or treatment with a chemical oxidizing agent, or a process which is plasma treatment, sodium hydroxide solution treatment, hydrophilic electroneutral polymer modification, adsorption of surfactants and/or influence or modification of the surface charge or the degree of particle wettability.

10. A mucoadhesive delivery system comprising:

a) a matrix comprising at least one substance and/or active pharmaceutical ingredient (API); and

b) a mucoadhesive (or adhesive means) capable of adhering the system to a mucosa.

11. A system according to claim 10 wherein:- a) the matrix comprises a nanoscaffold, preferably comprising biocompatible polymer(s), and optionally having pores of from 10 nm to 1000 μιη;

b) the mucoadhesive is a layer or adhesive portion and/or is capable of, or adapted to, secure, attach or adhere the system to a mucosa;

c) the mucoadhesive layer (at least in one part thereof) overlaps the matrix, so as to expose part of the layer to the mucosa;

d) the substance and/or API is in the form of, or comprised in, particles;

e) the nanoscaffold has a high surface area, high density of concentration of the API and/or is hyperporous; and/or

f) the nanoscaffold comprises biocompatible or natural polymers (that are nanofibers) that are highly cross-linked and/or electrospun or have been produced by electrospinning, for example naturally occurring polymers such as a peptide, carbohydrate, saccharide or polyamide.

12. A system according to claim 11 or 12 wherein:

a) the matrix comprises nanofibres comprising fibroin, such as silk fibroin;

b) the nanofibres have a water contact angle of at least 10 degrees but less than 60 degrees; c) there is primarily hydrophilic or H- bonding between the nanofibres and the nanoparticles; d) the prime interactions between the fibres and nanoparticles are polar, charged and/or electrostatic; and/or

e) the nanofibres comprise a polypetide that can form beta-sheets.

13. A method for preparing a mucoadhesive carrier or system according to any one of the preceding claims, characterized in that a nanoscaffold is joined or contacted with a mucoadhesive layer and/or a cover layer, and preferably an intermediate layer is inserted between said layers (usually before joining the layers).

14. The method according to claim 13, characterized in that the mucoadhesive layer (suitably prepared by any (industrial) method, e.g. by solvent casting method) and/or the intermediate layer (such as in the form of polymeric film layer or nanoscaffold) and/or the cover layer (preferably prepared in the form of polymeric film layer) is formed separately, the nanoscafold (layer) preferably prepared by an electrostatic spinning method (e.g. in the form of nanofibres), and optionally then the layers are firmly joined in the desired order, and preferably the nanoscaffold is prepared in situ onto the mucoadhesive layer and/or the cover layer and/or the intermediate layer.

15. The method according to claim 13 or 14, characterized in that a substance and optionally at least one excipient is deposited onto the nanoscaffold, either after its preparation or after joining all layers of the mucoadhesive carrier, preferably said substance and/or excipient being in the form of a solution, colloid or suspension.

16. The method according to claim 15, characterized in that the mucoadhesive carrier with the substance is then lyophilized or dried.

17. The mucoadhesive carrier or system according to any one of claims 1 to 11, characterized in that it is adapted for administration manually or by (an applicator) device (suitably by pressure directly to the target mucosa) preferably so that the nanoscaffold faces or adheres to the mucosa.

18. The mucoadhesive carrier or a system according to any one of claims 1 to 11 for:

a) use as or with a vaccine, preferably for delivery to mucosal surface(s), especially for sublingual vaccination and/or immunotherapy; or

b) delivery of therapeutic particle(s) preferably with local and/or systemic effect.

19. A mucoadhesive carrier or system according to any of claims 1 to 11 for use in medicine or therapy and as, or in, a medicament or therapeutic.

20. A mucoadhesive carrier or system according to any of claims 1 to 11 for use in a method of treatment and/or diagnosis of the human or animal body.

21. A mucoadhesive carrier or system according to any of claims 1 to 11 in the manufacture of a medicament or vaccine for the prophylaxis or treatment of a disease or condition in a human or animal.

22. A naturally occurring or biocompatible polymer which has been subjected to electrospinning, or has been electrospun.

23. An electrospun polymer according to claim 22 which comprises a polypeptide, or has a water contact angle of greater than 10 degrees and less than 60 degrees and/or has random and/or multidirectional fibres.

24. An electrospun fiber according to claim 22 or 23 which comprises a polypeptide that can form beta-sheets and/or has a predominant or high amount of Ser, Ala and/or Gly polypeptides, for example fibroin (such as silk fibroin).

25. An electrospun nanofiber according to any of claims 21 to 24 which additionally comprises, or has attached thereto, nanoparticles.

26. A mucoadhesive carrier or system or a process for the preparation thereof, substantially as herein described with reference to the Examples and/or Figures/drawings.