A pharmaceutical formulation for the delivery of vaccines and other nucleic acid therapeutics using bilayer mucoadhesive film

Abstract

Pharmaceutical composition comprising a bilayer mucoadhesive film comprising at least two consecutive interconnected layers and incorporating at least one active agent selected from proteins, nucleic acids and combinations thereof, adapted for buccal administration. Said films for use as a medication, in particular for use in treating viral diseases. A method for preparing said film, comprising the steps: a) preparing a backing layer by spreading a liquid composition comprising a water-insoluble polymer, and allowing to dry at ambient conditions to obtain a dried layer; b) preparing a liquid mucoadhesive mixture containing at least one active agent selected from proteins, nucleic acids, and combinations thereof, pouring the mucoadhesive mixture onto the dried layer from step (a), and allowing to dry at ambient conditions to obtain a dried bilayer film; and c) cutting the dried bilayer film into the desired shape and size.

Description

[0001] A pharmaceutical formulation for the delivery of vaccines and other nucleic acid therapeutics using bilayer mucoadhesive film

[0002] FIELD OF THE INVENTION

[0003] The present invention provides a novel technology for the delivery of vaccines and other therapeutic nucleic acids, including mRNA packed in lipid nanoparticles (LNP) or viruses incorporated into the slowly dissolving, mucoadhesive bilayer films designed for buccal administration. The present disclosure outlines a possible application wherein mucoadhesive films are used as a booster dose in heterologous vaccination.

[0004] BACKGROUND OF THE INVENTION

[0005] Nucleic acid-based therapeutics are used in preventive and curative form for diverse diseases, such as cancer, genetic disease, or vaccines that are delivered by different delivery strategies, for example, viral vectors, electroporation of nucleic acids, or by using lipid nanoparticles (LNP). Many nucleic acid-based medical products have been registered and approved or are currently undergoing clinical trials, for the treatment and prevention of a wide range of diseases. These medical products include antisense oligonucleotides (ASO), messenger RNA (mRNA) delivered by LNPs, short interfering RNA (siRNA) and conjugates, and different viral vectors (adeno-associated viruses (AAV), lentiviruses (LV) and adenoviruses (AV)) [1], Nucleic acid-based therapeutics can be administered by various delivery routes, including subcutaneous, intramuscular, intrathecal, subretinal, intravenous, or intravitreous delivery. The final result of nucleic acid administration is often a translation of a therapeutic protein with long-lasting curative or preventive effects, gene inhibition, addition, replacement, editing, or induction of a lasting immune response [2], The most widely used nucleic acid therapeutics to date have been COVID-19 vaccines, such as LNP-delivered mRNA vaccines BNT162b2 and mRNA-1273, or adenovirus-vectored vaccine AZD1222 [3], Vaccines are typically administered by intramuscular injection, which provides a systemic effect. However, this route of administration may not be optimal for respiratory infections, where the upper respiratory tract represents the entry point for pathogens and where the immune response should be the strongest [4], Poor induction of mucosal immunity, constant viral mutations of some pathogens, and waning immunity are some of the factors contributing to inadequate immune protection. Importantly, a significant proportion of the population is hesitant to receive vaccines via injections, leading to lower vaccine uptake rates [5], This reluctance may be due to needle-related fear, concerns about vaccine safety, or other factors, representing a significant public health concern since insufficient vaccine uptake increases the risk of outbreaks of vaccine-preventable diseases. The shortcomings of parenteral vaccination strategies have increased the demand for alternative routes of delivery such as mucosal vaccines, which have emerged as a promising alternative to injectable vaccines [6], By bypassing the need for needles, noninvasive mucosal vaccines are expected to improve patient compliance and provide more effective and longer-lasting protection against a wide range of infectious diseases, particularly respiratory, sexually transmitted, and gastrointestinal infections [7], Overall, the implementation of mucosal vaccines represents an important advancement in the field of vaccination and has the potential to significantly improve public health outcomes.

[0006] An alternative delivery route through mucosal surfaces is intranasal delivery, wherein a small dose of antigen is directly delivered into the respiratory tract [8], However, it suffers from the uptake variability of the vaccine due to anatomical differences between individuals, the state of the nasal mucosa, and a fast clearance rate [9], An additional concern is the potential intranasal delivery of ingredients into the brain via olfactory nerves, which could potentially trigger inflammation in the brain if the vaccine contains or produces adjuvants, including nucleic acids themselves

[0010] [ll]

[0012] , Substances delivered through the nasal cavity are also rapidly absorbed and quickly distributed throughout the body, which may cause systemic toxicity. Furthermore, the application of a nasal vaccine can also induce local irritation or inflammation, leading to discomfort and adverse reactions.

[0007] In contrast, oral vaccines are the most convenient and widely used mucosal vaccines. These methods offer the advantage of self-administration, making them particularly appealing for large-scale vaccination initiatives. By eliminating the need for needles, oral vaccines are not only convenient but also contribute to reducing the overall costs of vaccination programs. Consequently, they play a crucial role in benefiting low- income countries, where resources may be limited. However, oral administration of vaccines can be associated with gastrointestinal degradation of the vaccine and potential interference with the normal microbiota

[0013] , An optimal oral delivery route would be one which would maintain the slow release of the vaccine delivered to the head lymph nodes and bypass the stomach environment and first-pass liver metabolism. In this case, antigens would be directly delivered to the respiratory mucosa, which contains numerous lymph nodes and a dense capillary vessel network crucial for adequate immune response. This can be achieved by using mucoadhesive films that adhere to the oral mucosa and slowly release active ingredients, which penetrate through the non-keratinized buccal mucosa, where drugs are easily absorbed

[0014] , which also decreases vaccine loss through the gastrointestinal tract. Mucoadhesive films have been successfully employed for the delivery of small molecules but not for larger active ingredients such as vaccines that comprise protein antigens or nucleic acids in the form of viruses or mRNA formulated in LNPs. Previous studies have reported the suitability of mucoadhesive films for various applications, including food supplements, local anesthetics, and breath fresheners. Other buccal liposomal dosage forms that have been previously described include silymarin liposomes

[0015] , buccal micellar insulin spray, and buccal deformable liposomes to deliver insulin

[0016] , So far, mucosal vaccine delivery was only tested on non-user- friendly options such as transepithelial needle injections or gene gun-mediated injections

[0017]

[0018] ,

[0008] In this invention, we introduce bilayer mucoadhesive films as the delivery vehicle for vaccine delivery and delivery of other nucleic acid therapeutics. Polymers used for mucoadhesive film formation determine adhesive properties, biocompatibility, and drug release properties. The appropriate combination of polymers and their respective ratios can modify the dissolution profile of films, allowing for either rapid dissolution or a controlled release. As antigens used in vaccinations are typically water-soluble, we limited our evaluation to hydrophilic polymers. Our findings suggest that the solution of pullulan, trehalose, and sucrose forms films with highly desirable properties, such as a homogeneous appearance and excellent compatibility with incorporated antigens. To ensure targeted dissolution towards the mucosa, films are composed of at least two layers. The first mucoadhesive layer adheres to the mucosa, slowly dissolves, and releases active ingredients. The second, water-impermeable layer, prevents the active ingredient from dissolving in the oral cavity and increases the efficiency of absorbed active ingredients.

[0009] Previous studies have shown that a heterologous prime-boost approach is more effective than a homologous approach at inducing an immune response

[0019] , which can be further strengthened by inducing immune cross-protection using a combination of different routes of vaccine delivery, which can modulate magnitude and localization of immune response

[0020] , Combining mucosal vaccines and parenteral vaccines has been shown to induce a strong immune response at mucosal surfaces, including the respiratory, gastrointestinal, or genitourinary tract. This has been demonstrated with “Prime and Spike” parenteral priming and intranasal boosting

[0021] , When used in combination with pre-existing intramuscular prime vaccine, mucosal vaccines can elicit both systemic and local response, resulting in the recruitment of antigen- presenting cells and increased production of immunoglobulins A (IgA) by administration of mucosal vaccine and a systemic immunoglobulins G (IgG) by intramuscular vaccine. One of the critical components in fighting respiratory infections is the production of IgA, which is poorly induced using only an intramuscular vaccine regime. IgA plays a critical role in protecting the mucosal surfaces where infectious agents often invade the body. Immunoglobulins bind to pathogens and prevent them from attaching or penetrating the mucosal barrier. It has been reported that IgA activity diminishes quickly after receiving an intramuscular injection, and an intramuscular booster dose does not further enhance the IgA response

[0022] , Therefore, the administration of booster doses using the mucoadhesive-based vaccination strategy following intramuscular priming might be particularly effective. Mucosal vaccination is a highly effective strategy for simulating natural infection by activating innate and adaptive immune responses in the mucosa-associated lymphoid tissues. This approach is particularly beneficial for viruses such as Omicron, which persist in the upper respiratory tract and exhibit prolonged shedding

[0023] , Delivery of antigens through the oral tissues mimics natural infection inducing potent and durable immune memory in crucial infection sites such as tissue-resident memory T cells (TRM), B cells (BRM), and mucosal IgG and dimeric IgA

[0024] , In contrast, currently approved SARS- CoV-2 mRNA-LNP-based and vector-based vaccines, administered intramuscularly are less effective at inducing robust immune memory in these essential locations

[0025] , Implementation of mucosal vaccines is expected to improve patient compliance, and accessibility, and provide effective long-lasting protection against various infections. Oral vaccines can help reduce the risk of transmission of infectious diseases since they stimulate immunity at the mucosal surfaces of the body which serve as an entry portal for a wide range of pathogens

[0026] , The combination of secretory IgA and other immune cells induced by the heterologous regime, therefore, provides comprehensive protection against a wide range of pathogens. This type of formulation might be particularly convenient for the regular updating of vaccination, to maintain the immune level of the adapted vaccines against the prevailing variants.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention relates to a carrier system for the administration of antigens and other therapeutic nucleic acids via a buccal mucosal epithelium. The formulation utilizes a bilayer mucoadhesive film, which is applied onto the buccal mucosa, where it adheres and subsequently releases the incorporated antigens. Buccal mucosa was selected as the target site for vaccine administration, because of non-keratinized epithelium, which facilitates drug penetration. The main components of the film are natural and biodegradable polymers, which are ideal for creating customized films with specific release properties. Mucoadhesive films, therefore, afford flexibility in delivering different types of vaccine formulations, inducing an immune response, and providing a potentially non-invasive and user-friendly vaccination option. The bilayer composition of the film ensures a unidirectional release and prevents the loss of the incorporated vaccine into the oral cavity. The mucoadhesive layer is composed of trehalose, pullulan, and sucrose, which are compatible with commonly used vaccine types, namely proteins, nucleic acids, viral vectors, and mRNA-LNP. The waterinsoluble backing layer made of ethyl cellulose, protects the mucoadhesive layer and the incorporated antigens from saliva and enzymes present in the oral cavity. Furthermore, cargo permeation through the oral mucosa can be significantly improved by the addition of permeation enhancers. However, in the case of incorporated LNPs, uptake into the epithelial cells of the buccal mucosa occurs due to their lipophilicity. In this invention, we proved it is possible to pack the biologically active moiety into the film without interfering with its activity. When a mucoadhesive film is dissolving, the incorporated antigens are slowly released in their native state. This can be detected either in vitro by infecting cell cultures or by in vivo experiments conducted on animal models. Specific in vivo response is detected by luminescence in the case of the sequence coding for firefly luciferase or by induction of immune response in the case of antigens coding for the Spike protein or receptor binding domain (RBD) of SARS- CoV-2. After buccal immunization with vaccine-loaded bilayer films, animals showed comparable antigen-specific IgG titers to those immunized by intramuscular injection. Moreover, the mucosal response induced by this method was significantly higher compared to the one achieved through intramuscular injection. Antigen-specific antibodies, such as IgA made by plasma cells, are secreted to the mucosal surface, where they neutralize and eliminate pathogens, which is crucial for the body's defense against respiratory infections. In this invention, we introduced a user-friendly antigen delivery vehicle, where the incorporated antigens are protected from the degradation of the gastrointestinal tract and can induce local mucosal as well as systemic immunity. Furthermore, our findings suggest that this formulation has the potential to improve the conventional parenteral vaccination regime and could be used for vaccination strategies combining different routes of vaccine delivery.

[0012] DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention relates to a bilayer film, with good mucoadhesive properties that gradually dissolves upon placement on the buccal mucosa. A mucoadhesive layer of these films is designed to be compatible with the incorporation of various nucleic acids, such as plasmid DNA and mRNA in free form or packed in lipid nanoparticles or viral vectors. A mucoadhesive layer is composed of polymers and prepared using solvents, which do not affect the stability and activity of incorporated biologically active ingredients. This means the choice of buffers, pH, and other solvent characteristics are such that they do not adversely affect the incorporated antigens. A backing layer is prepared from any polymer or combination of polymers and other chemicals, which when dissolved in an appropriate solvent and cast form a firm film that is resistant to water and saliva, meaning it preserves its shape and composition even upon contact with moisture and that they are not toxic even if they are ingested. Notably, the backing layer seamlessly adheres to the mucoadhesive layer without any observable separation.

[0014] To ensure consistency, the same optimized formulation of the mucoadhesive layer and backing layer was used throughout all of the examples. The mucoadhesive layer was composed of 0,15 g of pullulan, 0,032 g sucrose, 0,032 g trehalose, and 0,037 g of glycerol per 1 ml of aqueous solution but could also be varied by up to fivefold for each ingredient. For easier detection, the films were engineered to incorporate isolated protein as the active ingredient for physical characterization, including the measurement of dissolution properties. To validate the concept, plasmid DNA or mRNA-LNP encoding firefly luciferase was incorporated into the films and placed onto murine mucosa to enable easier detection upon penetration of the oral tissue. To showcase the potential application of vaccination delivered by mucoadhesive films, films containing mRNA-LNPs and viral vectors were tested for their excellent penetration properties. In the following experiments, we demonstrated a potential vaccination approach toward Covid-19, which holds promising implications preferentially for addressing various respiratory infections. The following detailed examples are provided to offer a comprehensive illustration of the invention and its diverse applications. It is important to note that these examples do not restrict the scope of the invention but rather serve to enhance understanding of its implementation and potential uses for other vaccines or therapeutics to treat infectious diseases or other diseases where nucleic acids or proteins or their combinations are to be delivered into the upper respiratory tract and immune tissue and cells within the head.

[0015] According to a first aspect, the present invention provides a pharmaceutical composition comprising a bilayer mucoadhesive film comprising at least two consecutive interconnected layers and incorporating at least one active agent selected from proteins, nucleic acids and combinations thereof, adapted for buccal administration.

[0016] The bilayer mucoadhesive film comprises

[0017] (i) a first layer comprising the at least one active agent, wherein the first layer is adapted to adhere to buccal mucosa and release the at least one active agent into the mucosa, and (ii) a second layer, which is insoluble in water or saliva.

[0018] When applied to the mucosa, the first layer adheres to the mucosa while the second layer serves as a backing layer facing the oral cavity. It covers the first layer and prevents the at least one active agent from dissolving in the oral cavity.

[0019] The first layer typically comprises natural and biodegradable polymers with good bioadhesive properties and desired release properties. It slowly dissolves and releases the active agent(s). According to a preferred embodiment, the first layer comprises at least one hydrophilic polymer, in particular pullulan, trehalose and sucrose. Pullulan can be present in an amount in the range of 5-20%, more preferably 15% w / v, trehalose can be present in an amount of 1 -5%, more preferably 3.2% w / v, and sucrose can be present in an amount of 0.5- 10%, more preferably 3.2% w / v. Optionally, additional components can be present in the first layer, for example additives such as plasticizers and penetration enhancers to improve the physical properties and penetrability of the incorporated active agent(s). For example, glycerol can be used to enhance the spreadability, flexibility and elasticity of the film.

[0020] The first layer preferably has a thickness in the range of 200-800 pm, in particular 300- 500 pm, more preferably about 400 pm. A thicker mucoadhesive layer can increase the retention time of the film and ensures higher amounts of incorporated biologically active agents. However, if the layer is too thick, the film might be more prone to detachment from the mucosa.

[0021] To ensure the unidirectional release of the active agent(s) towards the buccal mucosa, the side of the mucoadhesive film facing the mouth cavity is layered with a protective and water-insoluble second layer (backing layer). The second layer comprises or consists of at least one water-insoluble polymer, in particular cellulose or cellulose derivative(s), preferably ethyl cellulose. It serves as a protective barrier to reduce the surface not in contact with the mucosa. This ensures that the cargo is released only in the direction of the mucosal surface and not into the surrounding tissues or fluids. Typically, the second layer is thinner than the mucoadhesive layer. It may have a thickness in the range of 20-50 pm, more preferably about 30 pm. The active agent may comprise at least one nucleic acid, such as those selected from antisense-oligonucleotides, double- or single-stranded DNA, RNA, in particular mRNA, or siRNA, and combinations thereof. Nucleic acids can be present in free form or packed, e.g. in lipid nanoparticles or viral vectors. According to a particular embodiment, the at least one nucleic acid is packed in a viral vector preferably selected from adenovirus, Modified Vaccinia Ancara (MVA), and lentivirus.

[0022] A pharmaceutical composition of the invention can be adapted to deliver the active agent(s) into the upper respiratory tract.

[0023] A particularly advantageous aspect of the invention is a pharmaceutical composition described herein which is storage stable, i.e. the bilayer mucoadhesive film remains stable for up to one week or even longer when stored in a refrigerator.

[0024] According to the invention, the pharmaceutical composition can be a therapeutic composition or a vaccine. It can be used in the prevention or treatment of various medical conditions and diseases such as viral infections, e.g. Covid19. For example, a vaccine based on a pharmaceutical composition of the invention can be for use in preventing a viral disease, in particular Covid19.

[0025] Another embodiment of the invention relates to a method for delivering biologically active agents selected from nucleic acid, proteins and combinations thereof using bilayer mucoadhesive films, wherein the mucoadhesive film is placed on the nonkeratinized epithelium of buccal mucosa, allowing for slow dissolution of the film and directed slow release of active ingredients. The bilayer mucoadhesive film can be a pharmaceutical composition as described herein.

[0026] According to preferred embodiments, the active agents can be any therapeutic proteins or nucleic acids, or antigens capable of eliciting an immune response in a subject.

[0027] When the at least one active agent contained in the bilayer mucoadhesive film is an antigen, the pharmaceutical composition according to the invention can be used to elicit an immune response. Thus, another aspect of the invention is a method for eliciting a specific immune response, preferably both a systemic immune response and a local immune response by delivering an antigen through the oral mucosa using a bilayer mucoadhesive film as described herein. For the delivery of a protein antigen, it is preferred that the antigen is released in its native state during film dissolution.

[0028] Another aspect of the invention relates to a method for preparing a pharmaceutical composition as described herein, comprising the steps: a) preparing a backing layer by spreading a liquid composition comprising a waterinsoluble polymer, in particular ethyl cellulose, preferably at a thickness of the liquid composition in the range of 500-2500 pm, in particular 1000-2000 pm, more preferably about 1500 pm, and allowing to dry at ambient conditions, preferably for 10-24h, in particular 12-18h, more preferably about 16h, to obtain a dried layer; b) preparing a liquid mucoadhesive mixture containing at least one active agent selected from proteins, nucleic acids and combinations thereof and one or more hydrophilic polymers, pouring the mucoadhesive mixture onto the dried layer from step (a), preferably at a thickness of the liquid mixture in the range of 1000-4000 pm, in particular 2000-3000 pm, more preferably about 2500 pm and allowing to dry at ambient conditions for preferably for another 10-24h, in particular 12-18h, more preferably about 16h, to obtain a dried bilayer film; and c) cutting the dried bilayer film into the desired shape.

[0029] The liquid composition comprising a water-insoluble polymer can be a solution or dispersion, such as an aqueous dispersion comprising the water-insoluble polymer or a solution of the water-insoluble polymer, e.g. in alcohol or acetone or a mixture thereof, such as a mixture or acetone and 2-propanol. The liquid composition may optionally contain additional components such as dibutyl phthalate.

[0030] In step (a), the liquid composition is preferably poured onto a release liner using a film applicator. The concentration in the liquid composition and the thickness of the wet layer can be adapted to obtain the desired layer thickness.

[0031] The mucoadhesive mixture can be an aqueous solution comprising the active agent(s) and one or more hydrophilic polymers and optionally one or more additional components. EXAMPLES

[0032] Example 1 : Preparation of bilayer mucoadhesive films.

[0033] Mucoadhesive films adhere to mucous membranes and improve drug absorption and bioavailability by providing longer retention times at the site of application through adhesion to the mucous membranes. These films are typically composed of natural and biodegradable polymers with good bioadhesive properties and desired release properties. The mucoadhesive film should be flexible, not too brittle, and durable enough to withstand mechanical stress and handling. Moreover, the incorporation of additives, such as plasticizers and penetration enhancers, can significantly improve the physical properties and penetrability of the incorporated antigens. However, it is crucial to ensure that all of the film-forming components received regulatory approval for safe human consumption. Additionally, the compatibility of the film components and delivered cargo should be carefully evaluated. The selection of polymers and other constituent elements typically guides the manufacturing process. To ensure the unidirectional release of the antigen towards the buccal mucosa, the side of the film facing the mouth cavity is layered with a protective and water-insoluble layer. This ensures that the cargo is released only in the direction of the mucosal surface and not into the surrounding tissues or fluids (Figure 1).

[0034] Results: Mucoadhesive films used in the described experiments were prepared from pullulan (15% w / v) (Polyscience), D-(+)-Trehalose dihydrate (Sigma-Aldrich) (3,2% w / v), and sucrose (3,2% w / v) (Sigma-Aldrich) dissolved in PBS. The right combination and ratio of polymers, after the drying process, formed a homogenous, flexible film with good mucosal adherence, compatible with the incorporated antigens. The mucoadhesive layer was formulated using a solvent-casting method

[0027] . To enhance the spreadability, flexibility, and elasticity of the film, glycerol (3% w / v) was added to the final mixture

[0028] , All components were pre-mixed in water or an aqueous solution containing the selected proteins or nucleic acids and magnetically stirred for at least 2 hours at 150 rpm until a thorough homogenization was achieved. The resulting solution was covered and left in a fridge without stirring for one hour to ensure there were no air bubbles in the mixture. To prepare the backing layer, a mixture of 0.5 g of ethyl cellulose (30-70 mPa.s) and 0.1 g of dibutyl phthalate per 10 ml of a mixture of acetone and 2-propanol (2:1 ) was used. The ethyl cellulose mixture was poured onto a release liner using a film applicator, creating a layer of the desired thickness. The wet mixture of the backing layer was set to a thickness of 1500 pm and left to dry overnight at ambient temperature. After the protective layer was dry (16 h, RT), the solution containing mucoadhesive polymers was cast over the backing layer with a wet thickness set to 2500 pm. The cast film was dried for another 16 h at room temperature. The final manufacturing step involved cutting the mucoadhesive film sheet into individual, single-dose units. For animal immunization studies we used circular bilayer films (4 mm diameter), cut using biopsy punches (Vital Medical Supply). Films intended for human use were cut into rectangular shapes measuring 2x3 cm. This step ensured that the film can be conveniently and accurately administered to patients and enabled the precise control of drug dosage. Prepared patches were subsequently packed into individual packages made of metalized polyester or aluminum or stored in a petri dish until further use. Proper packaging and labeling further ensure the film's stability, efficacy, and safe use by patients.

[0035] Bilayer film cross-section morphology was evaluated by scanning electron microscopy (SEM), where both layers were smooth and clearly seen. SEM pictures indicated both layers are homogenous and densely packed. The resulting films had a homogenous appearance with smooth surfaces, showing no signs of defects or layer separation (Figure 1). The mucoadhesive layer of the film had an average thickness of 400 pm, while the backing layer had an average thickness of 30 pm. A thicker mucoadhesive layer can increase the retention time of the film and ensures higher amounts of incorporated biologically active ingredients. However, if the layer is too thick, the film might be more prone to detachment from the mucosa. The backing layer, on the other hand, serves as a protective barrier to reduce the surface not in contact with the mucosa, preventing the biologically active ingredients from being released to unintended areas and is typically thinner than the mucoadhesive layer. Example 2: Comparison of dissolution profiles for mucoadhesive films with and without a backing layer.

[0036] The addition of a backing layer provides a unidirectional release of biologically active ingredients by acting as a barrier that prevents the dissolution and release of the active ingredients into the oral cavity. This allows the cargo to be released only in the direction of the mucosal surface. The backing layer is typically made of a waterinsoluble material. Its addition has a substantial impact on the amount of time a film is attached to the mucosal surface, thereby improving the level of cargo penetration. The dissolution time of films with a backing layer and films without a backing layer was evaluated using a dissolution device, which is composed of a flow-through cell with two chambers separated by a membrane. In the donor chamber, a film was placed and firmly fixed with the edges of the flow cell. A custom-made, constant leveling device provided a steady hydrostatic pressure to the flow cell. The dissolution media, simulating saliva (0,1 M phosphate buffer, pH 5,8) was drained through the donor chamber and the acceptor chamber, resulting in the dissolution of the film

[0029] , In the dissolution studies, the films incorporating red fluorescent protein (RFP) were examined by measuring fluorescence at an excitation wavelength of 542 nm and emission at 604 nm, allowing straightforward detection. On average, each film with a weight of 150 mg (3 replicates per group) contained approximately 2 mg of isolated red fluorescence protein. We compared the in vitro release profile of the films containing a backing layer and those without. The eluted media from the system was sampled and analyzed with BCA and a fluorimeter.

[0037] Results: The in vitro protein release profiles of films with the addition of a backing layer and films without a backing layer are shown in Figure 2. The presence of a backing layer in mucoadhesive films had a significant impact on the release profile and time of dissolution. In the case of films without a backing layer, water penetrated the film from both sides, resulting in a faster release rate of the protein. However, bilayered films hydrated only on one side, which resulted in a slower dissolution rate and gradual release of the active ingredient. As a result, the film remained less hydrated, slowing down the protein release rate and prolonging the protein's presence at the site of administration and particularly the share of active ingredient released to the mucosa. Films without a backing layer released almost 100% of RFP in 30 minutes, whereas films with the addition of a backing layer released more than 80% of RFP in 90 minutes. To test whether the backing layer prevents cargo release to the oral cavity, we turned the bilayer film on the opposite side (inverted) to confirm the film's impermeability. In this case, the film released around 10% of the RFP in the first few minutes due to the film's overhangs that stick outside the flow cell. After this initial release, the amount of released protein remained the same even after an hour. Therefore, we concluded that the backing layer made of ethyl cellulose does not leak and is an essential part of the pharmaceutical formulation for the delivery of nucleic acids or proteins to the mucosa. It is also possible to add additional layers, such as the protective layer at the top of a mucoadhesive layer that can be removed before application to the mucosa, which can facilitate the application to prevent mucoadhesive layer from sticking to other surfaces and decrease desiccation during long term storage. Also, additional mucoadhesive layer may be added to contain enhancers of mucosal absorption to prime the mucosa and prevent destabilization of biologically active ingredients, such as LNPs or enveloped viruses.

[0038] Example 3: Preparation of mucoadhesive films containing nucleic acids (plasmid DNA) and their detection in the oral mucosal tissue of BALB / c mice.

[0039] In the process of evaluating the efficacy of the vaccine delivery system, it was essential to detect the delivery of nucleic acids in vivo. To facilitate easy detection, a bioluminescent reporter based on a DNA sequence encoding firefly luciferase has been chosen. Films were prepared following the same protocol as described in Example 1, with isolated and purified plasmid DNA. Because the success of vaccine delivery through mucoadhesive films largely depends on the ability of the drug to permeate the mucosal barrier, it was necessary to add penetration enhancers to improve drug permeability. Penetration enhancers and other surface active ingredients create transcellular perturbations, facilitating the penetration of active ingredients through minor changes in the oral epithelium. However, the mucosal epithelium has a short turnover life, which quickly restores its integrity, making these changes temporary. To explore the potential of mucosal delivery of plasmid DNA, films containing approximately 40 micrograms of plasmid DNA were applied to the buccal mucosa of BALB / c mice. Films were attached to the oral mucosa of anesthetized mice for one hour. Twenty-four hours later, bioluminescence was determined using IVIS III (Perkin Elmer) after the addition of D-luciferin (500 pg / ml). The signal intensity was quantified using Living Image software (Perkin Elmer, Waltham, MA, USA).

[0040] Results: Expression of firefly luciferase in the animal model serves as proof of the efficient penetration of plasmid DNA through the mucosa using the pharmaceutical formulations described in this invention. The highest expression was detected in the tissues surrounding the oral cavity. Films containing different permeation enhancers were used to deliver the plasmid DNA coding for firefly luciferase to the oral mucosa. We tested Tween 80 and sodium tauroglycocholate (STGC), which were proven to be efficient penetration enhancers in mucoadhesive gels

[0030] , In our formulation, 1 % Tween 80 was found to be more effective than STGC in enhancing permeation, as evident by significantly higher levels of luciferase expression (Figure 3). Therefore, in subsequent experiments, 1 % Tween 80 was included in the films for the mucosal delivery of plasmid DNA or recombinant proteins. Tween 80 as a cell permeation enhancer acts on membranes and is as such not appropriate for the delivery of cargo in lipid vesicles or enveloped viruses but is appropriate for the delivery of proteins or nucleic acids in the concentration range of approximately 0.1 to 2%, preferably 1 %. This innovation is however not limited to Tween 80 but also compounds with similar action on plasma membranes, such as detergents. Further, Tween 80 could be implemented for the delivery also on proteins or nucleic acids in lipid-based particles for example by sequestration of permeability enhancers into an additional layer in the film, while the cargo could be packed into another layer, in order to allow enhancement of mucosal cell permeability with maintained integrity of the biological or therapeutically active cargo.

[0041] Example 4: In vivo and in vitro delivery of viral vectors encoding GFP via mucoadhesive films and assessment of their storage stability. Viral vectors have emerged as a promising platform for gene therapy as well as for vaccine delivery. Viral vector-based vaccines induce high immunogenicity and a robust cytotoxic T lymphocyte (CTL) response and have high efficiency for gene transduction and specific delivery of genes or antigens to target cells or tissues. In this example, we used two viral vectors known for their high immunogenicity, namely adenovirus (AV) and Modified Vaccinia Ankara (MVA) but could also be used for other viruses. Films were prepared as described in Example 1, using the same ratio of polymers dissolved in water containing viral vectors (AV or MVA). Before in vivo experiments, we performed preliminary tests on mammalian cell lines to evaluate the ability of the films to deliver packed cargo. For this experiment, we used HEK293 cells cultured in DMEM supplemented with 10% FBS. We prepared films containing adenovirus (AV) and films containing Modified Vaccinia Ankara (MVA), both encoding green fluorescent protein (GFP). Films with a diameter of 4 mm, were added directly to the cell culture medium of seeded cells (12 well plates), where they slowly dissolved, resulting in cell infection by viral particles incorporated in the film. After twenty-four hours, cells were harvested. As a control, we transfected the cells with plasmid DNA encoding GFP using a transfection agent. Furthermore, we used those films to infect female 8-10 weeks old BALB / c OlaHsd mice (Harlan Envigo, Italy). Animal studies were approved by government institutions, taking into account ethical considerations. The laboratory animals were housed in IVC cages (Techniplast) and provided standard chow (Mucedola) and tap water ad libitum. The mice were maintained in a 12-12 hour dark-light cycle. All animals used in the study were healthy and accompanied by health certificates from the animal vendor. Before the application of the mucoadhesive films, all animals were anesthetized and remained under anesthesia for one hour. One film with a diameter of 4 mm was applied to the buccal mucosa of each mouse. We used blank mucoadhesive films without viral vectors as the negative control. Twenty-four hours after film administration animals were subjected to fluorescence imaging. To evaluate the storage stability of the films containing viral vectors, we stored them at 4°C and room temperature for 7 days.

[0042] Results: Our findings confirm that functional viral particles are released from the polymeric matrix of the film and can infect the mammalian cells upon dissolution of the film in the cell medium. It demonstrates that the viral vectors can maintain their activity when packed in mucoadhesive film. In vivo, bioimaging of GFP expression was performed twenty-four hours after the application of the film. The highest expression of GFP was observed in the local area around the mouth cavity, indicating successful delivery to the oral mucosa. We also conducted a storage stability study by storing films containing viral vectors at both 4°C and room temperature for 7 days, followed by a repetition of all experiments. Our results demonstrate that the viral cargo integrated into the films remained stable for at least one week when refrigerated (4°C), indicating the potential for long-term storage and transport. These findings have important implications for the development of effective delivery systems. The AV viral particles retained their infection ability even after being stored at room temperature for one week. However, films containing MVA-based viral vectors stored at room temperature gradually lost some of their ability to infect cells or animals. Our experiments demonstrated that the formulation ensures infectivity of the viral vectors for up to 7 days at 4°C and up to some degree on RT, depending on which type of viral vector is used (Figure 4).

[0043] Example 5: BALB / c mice immunization with mucoadhesive films packed with AV-based viral vectors encoding RBD-bann.

[0044] To test the immunogenicity of delivered viral vectors via mucoadhesive films, we prepared films as described in Example 1, using the same ratio of polymers dissolved in water containing viral vectors. For immunization studies, we used an AV-based viral vector encoding the gene for receptor binding domain (RBD) of Spike protein from SARS-CoV-2 fused to the beta annulus peptide

[0031] , Fusion to beta annulus has been shown to result in high levels of neutralizing antibody production and potent viral neutralization upon immunization. To assess the immunogenicity of the mucoadhesive films containing viral vectors, we placed them onto the buccal mucosa of BALB / c mice (Harlan Envigo, Italy). Then we evaluated whether the viral vectors packed in the delivery vehicle could induce an antigen-specific humoral immune response. Animals were immunized with mucoadhesive films twice with a three-week interval between the doses, receiving the same formulation of antigen. Three weeks after the booster dose, blood was drawn and serum RBD-specific IgG titer was determined by ELISA (as described in Example 7). Results: Total RBD-specific IgG titer profiles of immunized mice are shown in Figure 5. Results suggest that mucosal immunization through mucoadhesive films induced RBD-specific IgG endpoint titers within three weeks after a single vaccination with AV- RBDbann. After receiving a booster dose, the IgG titers were found to be significantly higher in comparison to a control group. We concluded that immunization with AV- RBDbann via mucoadhesive films generates robust antibody response, supporting the further development of SARS-CoV-2-based mucosal vaccines composed of the bilayer mucoadhesive film and AV-based particles coding for selected antigen.

[0045] Example 6: In vivo delivery of LNPs mRNA coding for firefly luciferase delivered by mucoadhesive films.

[0046] Lipid nanoparticles (LNPs) have emerged as a promising vaccine delivery system due to their ability to encapsulate and deliver nucleic acid-based vaccines like mRNA. High efficacy rates of LNP-based mRNA COVID-19 vaccines developed by Pfizer / BioNTech and Moderna highlight the potential of LNPs as a vaccine delivery system

[0032] , LNPs also offer several other advantages, such as biocompatibility, biodegradability, and low toxicity. Due to their lipophilic nature, they can easily penetrate tissues and enhance drug absorption at the targeted site without the need for penetration enhancers. Mucoadhesive films containing mRNA-LNP were prepared following the same protocol as described in Example 1. The integrity of LNPs in the film was confirmed with dynamic light scattering (DLS). Films containing mRNA-LNPs encoding firefly luciferase were used to infect the HEK293 cell line. Films were dissolved in the medium of seeded HEK293 cells (12 well plates), where mRNA-LNPs were released from the film and infected mammalian cells. We confirmed the transfer of the functional mRNA by measuring luciferase activity in HEK293 cells. Moreover, we tested the effectiveness of films for in vivo delivery, by placing the mRNA-LNP- containing films on the buccal mucosa of BALB / c mice. Twenty-four hours after film administration the response was detected by measuring luciferase activity. Bioluminescence was determined using MS III (Perkin Elmer) after the addition of D- luciferin (500 pg / ml). Signal intensity was quantified using Living Image software (Perkin Elmer, Waltham, MA, USA Results: By measuring luciferase activity, we confirmed that films release mRNA- LNPs that can transfect cells and BALB / c mice without the use of additional penetration enhancers or adjuvants. We confirmed that their activity is maintained even after one month of storage and remained comparable to freshly made films. Twenty-four hours after film application on the murine mucosa the highest expression of firefly luciferase was detected locally, around the oral cavity (Figure 6) rather than in the gastrointestinal tract as shown previously for oral delivery.

[0047] Example 7: Heterologous prime-boost) vaccination regime with mRNA-LNP encoding Spike protein.

[0048] To evaluate the effectiveness of vaccination through the oral mucosa, BALB / c mice were immunized using mRNA-LNP vaccine three times at three-week intervals. The vaccination regimen consisted of an initial prime dose delivered intramuscularly, followed by booster vaccinations administered using mucoadhesive films. The scheme of heterologous immunization is illustrated in Figure 7. We expected that the parenteral prime dose would induce systemic virus-specific IgG response while the mucosal vaccine encoding Spike protein from SARS-CoV-2 given via mucosal routes will induce the secretion of specific IgA, which would provide additional protection against infection through the respiratory tract. To test the immunogenicity female 8-10 weeks BALB / c OlaHsd mice (Harlan Envigo, Italy) were immunized with a formulation containing mRNA-LNP encoding full-length Spike from SARS-CoV-2. Animals received a prime dose of mRNA-LNPs formulation via intramuscular injection, followed by a booster vaccination delivered with a mucoadhesive film in a three-week interval. For comparison, a group of animals received both prime and boost through mucoadhesive films and a negative control group received only blank films. mRNA- LNP vaccine was prepared by encapsulation of in vitro prepared capped mRNA encapsulated into LNPs in a microfluidic system. All animal studies were approved by appropriate government institutions, taking into account all ethical considerations. The laboratory animals were housed in IVC cages (Techniplast), and provided with standard chow (Mucedola) and tap water ad libitum. The mice were maintained in a 12-12 hour dark-light cycle. The animals used in the study were healthy and accompanied by health certificates from the animal vendor. The immunization was carried out under general inhalation anesthesia using 1.8% MAK isoflurane (Harvard Apparatus). The prime vaccine was administered using a 30G needle into the m. tibialis anterior after appropriate area preparation. The first boost was administered three weeks later. Animals were held under anesthesia when mucoadhesive films were applied to their buccal mucosa. To ensure that most of the mucoadhesive layer was dissolved they remained under anesthesia for 1 hour. Before receiving a booster dose, blood was drawn from the lateral tail vein using Microvette 300 (Sarstedt). Three weeks after the boost, the experiment was terminated, and the final blood sample was taken. The spleens, lungs, and brains of all animals were also harvested. Mouse sera were prepared by centrifugation of blood samples at 3000 rpm for 20 minutes at 4°C. The specific anti-Spike SARS CoV-2 total IgG in mouse sera was determined by ELISA to evaluate the immunogenicity of vaccines. Different classes of anti-Spike antibodies were also measured.

[0049] ELISA tests were performed to determine the endpoint titer. High-binding half-well plates (Greiner) were coated with the recombinant his-Spike SARS CoV-2 protein to determine specific anti-Spike SARS CoV-2 total IgG. The coated plates were incubated overnight at 4°C, washed with PBS+0.05% Tween 20 using an ELISA plate washer from Tecan, and then blocked for 1 hour at room temperature using ELISA diluent (PBS+3% FBS) solution. After washing the plates again, serial dilutions of mouse sera were added to the plates, with each dilution representing a certain titer value. The mouse sera were incubated overnight at 4°C. The plates were then washed, and goat anti-mouse IgG (H+L)-HRP antibodies (Jackson ImmunoResearch), diluted 1 :3000 in ELISA diluent solution, were added. The plates were incubated for 1 hour at room temperature, washed again, and then the TMB substrate was added. The reaction was stopped with the addition of acid solution (3M H3PO4) and measured absorbance at 450 nm and 620 nm by Synergy Mx microtiter plate reader (Biotek). Endpoint titers were calculated from measurements.

[0050] Results: The levels of Spike-specific antibodies were assessed in serum samples collected 21 days after each vaccination. Endpoint titers of Spike IgG antibodies in mouse sera were measured three weeks following the initial immunization and three weeks after the subsequent booster dose. Figure 8 illustrates the obtained results. Both groups receiving a vaccine (i.m.-film and film-film) exhibited antibody generation, with slightly higher titers observed in the group receiving an intramuscular prime dose. Notably, animals immunized with mucoadhesive films alone also demonstrated antibody production against the Spike protein, indicating their capacity to elicit an immune response. Generation of the most potent mucosal and systemic antibody response was obtained based on prior systemic priming. Based on these finding, we conclude that oral films represent an effective formulation of a booster vaccine and could be more effective in individuals who received multiple parenteral vaccine doses, as this approach leverages preexisting immunity and likely also in patients that have been preciously infected with the same virus. By administering mucosal vaccines, antigens initially come into contact with mucosal-associated lymphoid tissues, which play a pivotal role in inducing a mucosal immune response, which is crucial in combating sarbecoviruses and other respiratory viral infections. Furthermore, the vaccine formulation in this example did not include adjuvants, which are commonly associated with undesired effects such as inflammation when delivered through mucosal surfaces. However, the addition of an adjuvant would likely further potentiate the immune response, therefore also bilayer mucoadhesive film with biologically active nucleic acid or protein with added adjuvant is also one implementation of our invention.

[0051] Example 8: Mucoadhesive films are specifically designed to adhere to the oral mucosa, enabling controlled and localized delivery of therapeutic substances. This targeted delivery ensures that the immune response is focused on the oral mucosa, which is the desired site for stimulating mucosal immunity. Mucoadhesive films offer a superior alternative to intranasal application due to their ability to provide a targeted immune response without the risk of unintended delivery to the brain through the olfactory nerves. When administering vaccines and therapeutic agents through the nasal cavity, there is a concern about nucleic acids or antigens being transported directly to the brain via the trigeminal nerve, which can have undesirable consequences

[0033] , Intranasally delivered vaccines typically require powerful adjuvants like cholera toxin, which are commonly used to enhance the immune response but can also increase the likelihood of retrograde transport through the olfactory system, triggering the release of proinflam matory mediators. The effects of adjuvants in the hypothalamus after intranasal delivery may be attributed to their ability to enter the brain via retrograde transport along the olfactory nerve, bypassing the blood-brain barrier

[0034] ,

[0052] Furthermore, mucoadhesive films prevent the loss of non-mucosally absorbed drugs in the respiratory and digestive tracts. Unlike intranasal administration, where drugs can be easily cleared by mucociliary mechanisms

[0035] , mucoadhesive films adhere to the mucosa, ensuring prolonged contact time and improved drug absorption. This enhances the bioavailability and efficacy of the administered substances, leading to improved therapeutic outcomes.

[0053] FIGURES

[0054] Figure 1: Schematic illustration of a bilayer mucoadhesive film and cross- sectional scanning electron micrograph of a mucoadhesive film showing the composition and thickness of each layer. The bilayer mucoadhesive films are composed of two distinct layers: the mucoadhesive layer, which firmly attaches to the buccal mucosa, and the water-insoluble backing layer. The mucoadhesive layer has an average thickness of 400 pm (A) and the backing layer has an average thickness of 30 pm (B).

[0055] Figure 2: In vitro assessment of dissolution profiles of red fluorescence protein release from a bilayer film with the addition of a backing layer (o), a monolayer film without a backing layer (■), and an inverted film to evaluate impermeability of the backing layer (▼), using dissolution device.

[0056] Figure 3: Detection of a reporter gene luminescence in BALB / c mice using bilayer mucoadhesive films containing plasmid DNA coding for firefly luciferase and penetration enhancers. Through comparative evaluation, the impact of two penetration enhancers on luciferase expression is demonstrated. Each animal received one film containing 30 pg of plasmid DNA. Each dot represents an individual animal. No gel group is used as a control. Figure 4: In vivo delivery of MVA and AV encoding GFP through mucoadhesive films stored for one week at room temperature or one week at 4°C. Mucoadhesive films were placed onto BALB / c buccal mucosa for one hour. Twenty-four hours after administration of mucoadhesive films mice were subjected to fluorescence imaging. A) blank group receiving mucoadhesive films without viral vectors B) mice receiving mucoadhesive films containing GFP-MVA, after being stored for one week at room temperature C) mice receiving GFP-AV films stored for one week at room temperature D) delivery of GFP-MVA packed mucoadhesive films stored for one week at 4°C E) delivery of GFP-AV films stored for one week at 4°C.

[0057] Figure 5: Immunization of BALB / c mice with mucoadhesive films packed with AV-based vectors encoding RBD-bann of SARS-CoV-2. Three weeks after prime immunization, mice received a booster dose. Spike-specific serum IgG response was determined three weeks after the prime dose and three weeks after the booster dose. Statistically significant differences are indicated among groups receiving blank film without viral vector and a group receiving a mucoadhesive film packed with AV encoding RBD-bann. p values indicate significant differences ( **p < 0.01 ).

[0058] Figure 6: The expression of firefly luciferase detected in vivo after administering mucoadhesive films containing LNPs with mRNA encoding firefly luciferase onto the buccal mucosa of mice. BALB / c mice were subjected to bioluminescence imagining twenty-four hours after film administration. Luminescence intensity indicates the amount of expressed luciferase in the oral tissue.

[0059] Figure 7: Scheme of heterologous vaccination regime and time points of sample collection. BALB / c mice were immunized with an intramuscular prime application of mRNA-LNP vaccine encoding full-length SARS- CoV-2 spike protein, followed by two booster immunizations delivered via mucoadhesive film containing the same formulation of antigen, with a three-week interval between each dose. Blood was collected before each booster and at the time of termination, and analyzed for SARS- CoV-2 Spike-specific immunoglobulins. At the termination of the experiment, spleens were harvested for further experiments.

[0060] Figure 8: Endpoint titers of total IgG antibodies against SARS-CoV-2 Spike protein determined by ELISA three weeks after the prime dose (left) and three weeks after the booster dose (right). Each dot represents an individual animal.

[0061] DEFINITIONS

[0062] The term “vaccine” refers to the preparation introduced into the body of a human or animal that stimulates active acquired immunity to a particular infectious disease. The term vaccine as used herein refers to viral vectors and LNP containing mRNA, protein subunits, and plasmid DNA.

[0063] The term "antibody" as employed in this text refers to a defensive protein generated by B lymphocytes in reaction to the antigen. These classifications, in turn, define the different classes of immunoglobulins, namely IgG, IgM, IgA, IgD, and IgE.

[0064] The term “immune response”, as used herein, relates to an organ system in higher- developed organisms composed of specific cell subtypes, which act to eliminate foreign molecules from the organism.

[0065] The term “immunization” as used herein relates to the administration of prepared vaccine into a living organism or its body via oral route in the form of mucoadhesive film, which is placed on the buccal mucosa or by inhalation of intranasal spray or intramuscular inoculation via simple needle injection.

[0066] The term “prime and booster dose”, as used herein refers to the first dosage, usually administered intramuscularly and booster dose refers to any following administration of vaccine.

[0067] The term “Endpoint titer (EPT)” as used herein relates to ELISA-based or any other experimental determination and calculation of specific antibodies towards different antigens. The EPT, or endpoint titer, is defined as the highest dilution of serum that yields an absorbance value above the calculated cutoff value.

[0068] The term “lipid nanoparticles (LNP)” refers to delivery systems, as they can encapsulate and protect drugs or other therapeutic molecules such as mRNA and DNA, improving their stability and targeting their delivery to specific cells or tissues in the body.

[0069] The term “solvent casting” refers to one of the manufacturing techniques in the preparation of mucoadhesive films, where active pharmaceutical ingredients and other excipients are typically dissolved in a suitable solvent system forming a homogeneous mixture called "casting solution". The casting solution is then poured onto a casting sheet, where the solvent evaporates.

[0070] The term “bilayer mucoadhesive film”, as used herein refers to a thin, flexible sheet that is designed to adhere to the mucous membranes in the body f.e. mucosal surfaces in the mouth, throat, or gastrointestinal tract. They are commonly used as pharmaceutical delivery systems for drugs or other therapeutic agents. These films are made of a special type of polymers that can bind to the mucosa, creating a strong and long-lasting bond. By adhering to the mucous membranes, the films can provide sustained release of the medication, allowing it to be absorbed slowly and steadily into the bloodstream over a period of hours or even days. In our work, mucoadhesive films refer to films placed on the non-keratinized stratified squamous epithelium, which forms the outer buccal epithelium.

[0071] The term “buccal” refers to the anatomical part between the cheek and the gums, which is called the buccal endometrium. This is the place where drugs or vaccines enter into systemic circulation through jugular ducts via a network of blood vessels. It is considered the major barrier to the penetration of most drugs in buccal delivery. After the drug penetrates the buccal epithelium, it enters the systemic circulation via the vascularized tissue and jugular vein

[0072] The term “mucosa”, which is also known as mucous membrane, refers to a type of tissue that lines various cavities and surfaces in the body that are in contact with the external environment. It is composed of specialized cells that secrete mucus, a thick, viscous substance that helps to protect and lubricate the tissues.

[0073] The term “Spike” as used herein relates to the Spike glycoprotein of SARS-Cov-2 (UniProt: P0DTC2) is a protein on the surface of the virus that allows it to enter and infect human cells. The Spike protein binds to the ACE2 receptor in human cells, which initiates the process of viral entry and infection. The Spike protein is the primary target of many COVID-19 vaccines and therapeutics.

[0074] The term “mice” as used herein relates to BALB / c OlaHsd mice, an albino laboratory- bred strain, or any mice laboratory strain that is commonly used in immunological studies and immunization protocols.

[0075] The term “T cell”, as used herein, relates to lymphocytes, the T-subset (e.g. CD4+ and CD8+ T cells) of white blood cells, a specific mononuclear immune cell population that interacts in the adaptive immune system by recognizing antigen peptides bound to major histocompatibility complex (MHC) molecules with a T cell receptor.

[0076] This invention presents the use of mucoadhesive films as an efficient and safe immunization route for the mucosal delivery of vaccines, to provide an alternative approach to immunization or for the delivery of nucleic acid or protein therapeutics for other diseases. The presented examples demonstrate that viral vectors and LNPs, encoding the nucleic acids or antigen of interest, are well-suited for this formulation. We specifically selected viral vectors and mRNA-LNPs based on their ability to transfect cells and penetrate cell membranes, eliminating the need for penetration enhancers or other surface-active ingredients. These mucoadhesive films can be readily and quickly optimized or adjusted to ensure the delivery of the antigen for circulating viral strains.

[0077] To address the urgent need for widespread booster strategies with high acceptance and uptake rates, we propose integrating mucoadhesive films as an addition to the existing parenteral prime vaccine regime. This novel approach elicits both local and systemic immune responses, thereby enhancing the potential for comprehensive immune protection against respiratory viruses. By combining different vaccine regimens, we anticipate enhanced efficacy and protection against such pathogens. Our invention represents a significant advancement in vaccine and therapeutic protein or nucleic acid drug delivery, demonstrating the viability of delivering various antigens through mucoadhesive films.

[0078] It is important to note that our invention extends beyond the delivery of COVID-19 vaccines based on nucleic acids. The versatility of this technology enables its application to other nucleic acid treatments and prophylaxis. By providing an efficient and easy-to-implement platform for vaccine and biological drug delivery, our innovation also contributes to better compliance and improving the stability of vaccines and other pharmaceutical formulations.

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Claims

CLAIMS1. Pharmaceutical composition comprising a bilayer mucoadhesive film comprising at least two consecutive interconnected layers and incorporating at least one active agent selected from proteins, nucleic acids and combinations thereof, adapted for buccal administration.

2. The pharmaceutical composition according to claim 1 , wherein the bilayer mucoadhesive film comprises(i) a first layer comprising the at least one active agent, wherein the first layer is adapted to adhere to the buccal mucosa and release the at least one active agent into the mucosa, and(ii) a second layer, which is insoluble in water or saliva, wherein, when applied to the mucosa, the second layer faces the oral cavity and covers the first layer to prevent the at least one active agent from dissolving in the oral cavity.

3. The pharmaceutical composition according to claim 1 or 2, wherein the first layer comprises at least one hydrophilic polymer, in particular pullulan, trehalose and sucrose, preferably pullulan in an amount of range 5 - 20%, more preferably 15% w / v, trehalose in an amount of 1 - 5%, more preferably 3.2% w / v, and sucrose in an amount of 0.5 - 10%, more preferably 3.2% w / v.

4. The pharmaceutical composition according to any one of claims 1 -3, wherein the at least one active agent is not sensitive to detergents, and wherein the first layer comprises one or more enhancers of mucosal cell permeability, such as nonionic detergents, in particular Tween 80, preferably in a concentration in the range of 0.1 -2 % by weight based on the total weight of the first layer, preferably up to 1 %.

5. The pharmaceutical composition according to any one of claims 1 -4, wherein the second layer comprises or consists of at least one water-insoluble polymer, in particular cellulose or cellulose derivative(s), preferably ethyl cellulose.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the active agent comprises at least one nucleic acid selected from antisense- oligonucleotides, double- or single-stranded DNA, RNA, in particular mRNA, or siRNA, and combinations thereof, preferably in free form or packed in lipid nanoparticles or in viral vectors.

7. The pharmaceutical composition according to claim 6, wherein the at least one nucleic acid is packed in a viral vector preferably selected from adenovirus, Modified Vaccinia Ancara (MVA), Adeno-associated virus (AAV) and lentivirus.

8. The pharmaceutical composition according to any one of claims 1-7, which is adapted for delivery of the at least one active agent into the upper respiratory tract.

9. The pharmaceutical composition according to any one of claims 1-8, which is a vaccine or therapeutic composition.

10. The pharmaceutical composition according to any one of claims 1 -9 for use in the prevention or treatment of a disease.

11. The pharmaceutical composition for use according to claim 10, wherein the disease is a viral infection, in particular Covid19, or influenza.

12. A method for delivering biologically active agents selected from nucleic acid, proteins and combinations thereof using bilayer mucoadhesive films, wherein the mucoadhesive film is placed on the non-keratinized epithelium of buccal mucosa, allowing for slow dissolution of the film and directed slow release of active ingredients.

13. The method according to claim 12 for delivering a protein antigen to the oral mucosa, wherein the antigen is released in its native state during film dissolution.

14. A method for eliciting a specific systemic and local immune response by delivering an antigen through the oral mucosa using a bilayer mucoadhesive film, preferably a mucoadhesive film as defined in any one of claims 1 -9.

15. A method for preparing a pharmaceutical composition according to any one of claims 1 -9, comprising the steps: a) preparing a backing layer by spreading a liquid composition comprising a water-insoluble polymer, in particular ethyl cellulose, preferably at a thickness of the liquid composition in the range of 500-2500 pm, in particular 1000-2000 pm, more preferably about 1500 pm, and allowing to dry at ambient conditions, preferably for 10-24h, in particular 12-18h, more preferably about 16h, to obtain a dried layer; b) preparing a liquid mucoadhesive mixture containing at least one active agent selected from proteins, nucleic acids, and combinations thereof, pouring the mucoadhesive mixture onto the dried layer from step (a), preferably at a thickness of the liquid mixture in the range of 1000-4000 pm, in particular, 2000- 3000 pm, more preferably about 2500 pm and allowing to dry at ambient conditions for preferably for another 10-24h, in particular 12-18h, more preferably about 16h, to obtain a dried bilayer film; and c) cutting the dried bilayer film into the desired shape and size.

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