TECHNICAL FIELD

[001] The present disclosure relates to composition and devices for making barrier films on biological tissue.

DESCRIPTION OF RELATED ART

[002] Currently, physical barriers like masks are used to control invasion of pathogens in the human body. These masks create issues with efficiency, comfort and compliance. Moreover, masks are not complete-barrier on human face and not feasible in case of animals.

SUMMARY

[003] Methods, devices, kits, and compositions according to the present disclosure may be used to prepare barrier films, for example, that reduce or prevent passage of contaminants or pathogens.

[004] In embodiments, the present disclosure describes a barrier film against pathogens. The barrier film includes a gel. The gel includes a non-alginate polymer, a cross-linked alginate polymer and a microbicidal agent in the gel.

[005] In embodiments, the present disclosure describes a method for forming a barrier film against pathogens. The barrier film includes a gel. The method includes applying composition A on a biological surface, wherein the composition A includes a non-alginate polymer, a crosslinker, and a microbicidal agent. Further, the method includes applying a composition B on the biological surface, wherein the composition B includes sodium alginate. The gel is formed on the biological surface by a combination of the composition A and the composition B.

[006] In embodiments, the present disclosure describes a kit with a first container and a second container. The first container includes a composition A. The composition A includes a non alginate polymer, a crosslinker, and a microbicidal agent. The second container includes a composition B, wherein the composition B includes a sodium alginate.

[007] In embodiments, the present disclosure describes a dispensing device for forming a barrier film. The device includes a first enclosure defining a first volume and consists of composition A. The composition A includes a non-alginate polymer, crosslinker, and a microbicidal agent. The device includes a second enclosure defining a second volume and consists of composition B. The composition B includes sodium alginate. A first dispenser is coupled to the first enclosure and a second dispenser is coupled to the second enclosure. A first actuator is coupled to the first dispenser and the second actuator is coupled to a second dispenser. The first actuator is configured to cause composition A to be released from the first dispenser and the second actuator is configured to cause composition B to be released from the second dispenser.

[008] The details of one or more aspects are set forth in the accompanying drawings and description. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] FIG. 1 illustrates a barrier film on a biological tissue.

[010] FIG. 2. illustrates a kit with container first and second container

[011] FIG. 3 A illustrates a dispensing device for creating a barrier film wherein containers are connected, with the dispensers being on the same side.

[012] FIG. 3B illustrates a dispensing device for creating a barrier film wherein containers are connected, with the dispensers being on opposite sides.

[013] FIG. 4A illustrates a method of dispensing from the dispensing device wherein the composition A and composition B are added one after the other

[014] FIG. 4B illustrates a method of dispensing from the dispensing device wherein the composition A and composition B are dispensed so that both reach the barrier film at the same time.

[015] FIG. 5 illustrates a block diagram of method of formation of barrier film.

[016] FIG. 6 is a photograph showing the barrier film formed on the rubber teat

[017] FIG. 7 is a photograph showing microbial plates comparing positive and negative controls with three samples. DETAILED DESCRIPTION

[018] Methods, devices, kits, and compositions according to the present disclosure may be used to prepare barrier films, for example, that reduce or prevent passage of contaminants or pathogens.

[019] For example, barrier films according to the disclosure may reduce or prevent transmission, entry, or passage of pathogens or contaminants into or along tissues, from example, from an external environment. In some examples, the tissues may define body surfaces, for example, skin, mucous membrane, oral passage, or nasal passage. In embodiments, barrier films may be used instead of, or in addition to, other contaminant or pathogen reducing or preventing means, such as masks or respirators.

[020] Infections are caused by diverse organisms, viruses and bacteria are the most common pathogens. Infections in respiratory tract can occur either when the pathogen enters the respiratory tract through nasopharyngeal route or can be of blood-origin when due to systemic infections blood is carrying the pathogens. The nasal route of transmission has been regarded as predominant. The aerosol route is the most often indicated route since it has implications on the rapidity with which the pathogens can transmit within the community of people, stock of animals or flock of birds. From the point of view of understanding the mode of transmission of pathogens through aerosol route, the physical, aerodynamic and epidemiological behavior of aerosol-transmitted infections may be considered. In simple terms, when a patient suffering from respiratory infection may respire the pathogen as air-borne particles which, depending upon its size, has potential to be inspired and infect persons/animals in contact. A number of studies have shown that size of the air-borne suspended particle is a factor that influences pathogenicity. The Infectious Disease Society of America has defined “respirable particles” as having a diameter of 10 pm or less; and “inspirable particles” as having a diameter between 10 pm and 100 pm. Inspired particles, irrespective of the size, are first deposited in the upper airways, whereas, particles <10 um size rapidly reach the lower respiratory tract and lung alveoli.

[021] A person/animal suffering from respiratory infections produces infectious droplets of varying sizes by breathing, coughing or sneezing resulting expired air containing both short- range large droplets and airborne small droplet nuclei. Transmission of pathogens through airborne droplets depends on the distance from the pathogen source (sick animal or person). Transmission however is also possible physically by touching the nose area after coming in contact with the surface where larger particles have settled. Hence, the pathogens can be transmitted through nasal route irrespective of the particle size. Common diseases that can be transmitted through aerosol route are chicken pox, measles, tuberculosis, severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), influenza, Ebola in human. In case of domestic and pet animals, common pathogens transmitted through aerosol are bovine syncytial virus, avian influenza, anthrax, foot and mouth disease, swine flu, bovine/caprine pleuropneumonia, hemorrhagic septicemia etc.

[022] The virulent viruses have glycol-protein sites to attach to specific cells of the upper respiratory tract which initiates cascade of process of internalization called receptor-mediated endocytosis. Once inside the cell, the viral genome is replicated and transcribed to synthesize virion proteins, lipid membrane and anchoring of sugars to form virion particles. These virion particles are released from the infected cells leading to expanding of infection to more cells in the upper and lower respiratory tract. The resulting breaches in the respiratory epithelium results in an increased susceptibility to secondary infection. These new virions along with respiratory secretions form particles and are expired with coughing or sneezing infecting other in-contact susceptible person / animals. This process is more or less common to all the enveloped viruses such as herpes, retro, orthomyxo, paramyxo, flavi, Toga and Influenza viruses. The mechanism is more or less similar in non-enveloped viruses except that these lack a lipid membrane and may not have sugar residues. Once the body is heavily infected cardinal symptoms are expressed and if the host is able to mount immunological response, the virus multiplication stops and the person recovers.

[023] It is generally understood that the viral or bacterial diseases, especially the epidemics, would stop only when majority of the population develop immunity against the pathogen, which can be achieved by vaccinating large populations or when no vaccine is available, the population gets exposed to low dose of the pathogenic microbes.

[024] A similar pathogenesis route is followed by pathogenic bacteria. After entry from the natural routes, the pathogenic bacteria / viruses bind to the specific tissue receptors and then get internalized. Thus, binding of the pathogens to the tissues is an important event in pathogenesis and any loss of contact of the pathogen with the natural entry route tissues would result in no infection even after exposure. [025] Many viruses lack proofreading and error correction mechanisms in the virus transcriptional apparatus, and hence are prone to minor changes to one or both of the major surface antigens during replication. In such a case (which is more frequent) there is antigenic drift which leads to seasonal epidemics because the new variant of the virus can infect persons / animals having partial immunity from a prior exposure. For example, Influenza A viruses are especially prone to antigenic drift causing periodic epidemics leading to huge morbidity and mortality. The influenza virus is known to be cause of 20,000 to 40,000 deaths and up to 300,000 hospitalization in USA alone.

[026] In case of birds, Avian influenza is responsible to cause periodic epidemics throughout the world, leading to not only losses to the poultry industry but affecting damages to other sectors such as tourism, aviation and business. At present, 15 subtypes have been identified that can infect birds but only H7, H5 and H9 subtypes are associated with outbreaks. Viruses can also exchange or swap genetic material from other subtypes from other species and the mixing permits the virus to become virulent to new species. For these reasons vaccination to prevent the viral respiratory diseases are only partially successful.

[027] Currently, there are four antiviral drugs available in the market for treatment. Amantidine (Gocovir TM Adams Pharmaceuticals, Emeryville, CA, USA) is used as prophylactic in certain flu virus infections. It has several side effects such as bladder pain, confusion, dizziness, fainting, polyurea, swelling hands and feet. Development of resistance is another problem. Ramitidine (FlumadineTM ,Caraco Pharmaceutical Laboratories, Detroit, MI, USA) is also indicated in preventing certain types of flu virus infections. It also reduces severity of symptoms and helps in early recovery. It does not prevent infection but affects viral growth. There are side effects such as, nausea, vomiting, trouble sleeping, loss of appetite, vomiting, dry mouth, weakness and dizziness. Osaltamivir (Tamiflu TM , Hoffman La Roche, Basel, Switzerland) is another commonly used antiviral drug. It helps in reducing symptoms of flu and other viral infections. It must be taken with food to avoid upset stomach. The side effects include nausea and vomiting. Zanamivir (RelanzaTM , Adams Pharmaceuticals, Emeryville, CA, USA) is another antiviral drug indicated in flu infection. The drug has several side reflects, such as, headache, throat pain, nausea, vomiting, diarrhea, dizziness, nosebleed, eye redness or discomfort, sleep problems (insomnia). Oseltamivir as well as zanamivir are neuraminidase inhibitors that block the release of mature viral particles and thus prevent the infection of neighboring cells. Neuraminidase inhibitors lessen the symptoms of influenza infection and shorten the duration of the disease. But the limitation is that for effective prophylaxis the drug must be given within a 48 -hour window of the onset of symptoms and there is a risk of resistant strains emerging.

[028] Foot and Mouth Disease is an important highly contagious disease in domestic and wild animals. The major transmission is through nasal route. The disease causes heavy economic losses to the industry. Currently vaccination is the only strategy to control infection in in-contact animals but there is no strategy available to prevent transmission of the vims within the epidemic zone.

[029] Nosocomial infections affecting upper respiratory tract (URT) and lower respiratory tract (LRT) are very common in pet animals. The disease is caused by inhalation of pathogens in the veterinary clinic or in-patient environment. Similar nosocomial aerosol infections are also common in human patients hospitalized for a long duration. The CDC estimates two million people in the United States are infected annually by these infections resulting in 99,000 deaths as these infections are very difficult to treat and survivors remain chronically ill. Currently there is no solution available to prevent entry of these pathogens through the nasal route.

[030] Severe acute respiratory syndrome (SARS), Influenza and Corona vims infections in people and several respiratory syndromes in pet and domestic animals are now new global health challenges of the 21st century. Due to limitations in vaccination and treatment out-of-box solutions are required to control disease outbreaks.

[031] One plausible way to control the pathogen transmission is to block its attachment to the specific epithelial cells. For example, it has been shown that the Influenza vims after entering the respiratory tract binds to the a2,6-SA-bearing ciliated cells which are predominantly present in the upper respiratory tract, such as, nasal turbinate and pharynx, larynx), whereas its density in LRT respiratory tract is now high. Earlier it was thought that since coughing was assumed to transmit the infection, the source of exhaled vims could be lower respiratory tract, specially lung and bronchi. But recent in vivo studies have shown that SARs vimses are in fact transmitted via the air from the upper respiratory tract, more specifically from the nasal respiratory epithelium, and not from the trachea, bronchus or the lungs. Without being bound by theory, our hypothesis is that since the nasal epithelium plays a major role in acquiring new infection as well as transmitting the infection from a sick person to in contact-person(s), the novel solutions could be to block attachment of the vims / bacteria to the nasal epithelium by developing a novel barrier type formulation containing immobilized already-known microbicidal compounds and a sol-gel type excipient. Since such a strategy could also be useful for preventing transmission of bacteria, it will be possible to extend the concept of chemical or physical barrier film to prevent bacterial diseases entering the body through natural routes.

[032] Spraying into nasal cavity of pet animals and human coming to infected clinic and wards could be a plausible way to form a barrier film in the nasal cavity to prohibit entry of the nosocomial pathogens into URT.

[033] Wounds are a common problem in people and animals. A number of studies have shown that for wound healing the requirements are: (a) the wound should be insulated from the external contamination hence covered with dressing materials, (b) wound should always be moist so as to attract wound-healing cells and enhance metabolic activity leading to rapid healing and (c) microbicidal to take care of existing microbial contamination of the wound. In many cases, especially in animals, applying bandages or dressings is not feasible. The animals are left in open spaces hence drying of wound due to exposure to sun is a constraint and the animal barns are highly contaminated environments hence insulating wound from environmental pathogen is very difficult. These issues can be addressed by developing a formulation that after application could form a barrier film insulting the wound surface from environment, releasing moisture over a period of time and immobilized microbicidal to take care of any pathogen contaminating the wound but permitting aeration of the wound.

[034] Entry of pathogens from the environment and natural orifices like, teat canal is common in lactating animals. It is well known that after milking is over the teat orifice remains open for 1-2 hours or longer depending upon several host factors. During this period the lactating animal is at a very high risk of entry of skin or environment pathogens causing mastitis, reduction in milk yield and sometime loss of udder tissues. The disease causes significant economic losses to the farmers. To protect pathogen entry a common strategy is to dip each teat in an aqueous teat dip solution after milking, also called post-milking teat dip. A major disadvantage of the teat dip is that it comes off when the animal sits on wet floor. These solutions also do not withstand shear pressure of the animal’s activity. Few teat dips that claim barrier property are solvent based hence expensive and can cause severe harm to the skin since these solutions are used twice a day throughout the year. Our aqueous adhesive barrier film forming technology is a plausible solution. In small-hold dairy operations the barn hygiene standard may not be high hence due to bacterial overload on the barn floor the barrier film alone may not suffice. In such cases including contact microbicidal might be helpful. In case of lactating animals, presence of residual silver nano-particles may not be desired hence using thiolated chitosan with capped silver nano-particles might be more useful.

[035] Post-partum cervico-uterine infections are common in animals. The port of entry is cervix which remains open to entry of external pathogens for about 8-10 days and then closes once the process of involution starts. The barrier film technology can be used in such cases with the help of infusing of the two solutions intrauterine and in the cervix. Post-partum cervicitis and endometritis are common problems in women and animals. In women the problem is acute due to sub-hygienic practices in resource -poor women in developing and under-developed countries. In case of bovine and ovine females, however, it is a major gynecological disease- causing repeat breeding syndrome, secondary respiratory and mammary gland diseases, wherein the uterus becomes the primary infection source. The external pathogens have high potential of entry into uterus after birth of newborn because the cervix remains open for sometime and the uterus is immune-compromised. It has been proposed in cattle that cases of uterine bacterial contamination are directly associated with the aggravated disruption of the epithelium’s integrity, coupled by an influx of neutrophils and secretion of chemokines and cytokines, drawing the scenery of a persistent inflammatory response. The resulting endocrine dysfunction caused is ensued in the endometrium, presumably disrupting the embryo’s implantation.

[036] Another constraint is that repeated insertion of antibiotic solution or device is not without high infection risk if done in home / barn environment. Without wishing to be bound by theory, if the uterine mucous membrane is layered with a barrier film, the pathogens would not be able to attach to the uterine lining whereas once the cervix is closed due to physiological involution process, in the anerobic uterine environment the pathogens would not be able to survive. In embodiments, an intrauterine infusion solution that can form a barrier film over the uterine mucous membrane and also form a seal in the cervix so that contaminating pathogens in the uterus are not able to attach and form a nidus in the endometrium and further pathogens are barricaded due to the cervical seal. For example, the barrier film may be used to reduce or prevent vaginitis.

[037] The present disclosure describes devices, systems, kits, and techniques for forming coatings or barrier films against pathogens. For example, the coating can be adopted as spray or infusion that on administration would form a uniform film protecting the natural orifices, such as, nasal epithelium, teat canal, wound, cervix and uterus from binding of the pathogens, such as, bacteria / virus particles to the epithelial cells as well as prevent entry of bacteria due to contact microbicidal property and also due to size exclusion as the pore size of the resultant barrier film is less than 0.2 micron.

[038] FIG. 1 illustrates a barrier film on a biological tissue.FIG.l shows a system 10 with a barrier film 12 on a biological tissue 14.

[039] In embodiments, the present disclosure describes a barrier film 12 against pathogens. The barrier film includes a gel. The gel includes a non-alginate polymer, a cross-linked alginate polymer and a microbicidal agent in the gel.

[040] In embodiments, the non-alginate polymer can be a water-soluble biocompatible polymer. For example, the non-alginate polymer can be synthetic polymers like poly(ethylene glycol) (PEG), derivatives of PEG, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyacrylamides, N-(2-hydroxypropyl) methacrylamide (HPMA), divinyl ether-maleic anhydride (DIVEMA), polyoxazoline, polyphosphates or polyphosphazenes. Alternatively, the non-alginate polymer can be a natural water soluble polymer like xanthan gum, pectin, chitosan or chitosan derivatives, dextran, carrageenan, guar gum, cellulose ethers, hyaluronic acid, albumin, starch or starch based derivatives.

[041] In embodiments, the non-alginate polymer includes a chitosan polymer.Chitosan polymer isa natural polymer obtained by deacetylation of chitin. Chitosan is biocompatible, biodegradable and bioadhesive/mucoadhesive. Chitosan can also be modified to improve solubility over a range of pH. For example, carboxymethylation of chitosan results in formation of N- carboxymethylchitosan (N-CMC) which is soluble in wide range of pH. In embodiments, the chitosan is >75% deacetylated. In some embodiments, the chitosan has a molecular weight of 19000-375000 Da.

[042] In embodiments, the bioadhesive/mucoadhesive property of chitosan can be enhanced by modifying or grafting thiol to make thiolated chitosan. Chitosan can also be functionalized with polyethylene glycol (PEG) linkages to the reactive amino and hydroxyl groups of chitosan to make a PEGylated chitosan.

[043] In embodiments, the chitosan polymer can also include a crosslinker. The crosslinked chitosan will confer the ability to form a firmer and more effective barrier to protect biological surfaces. In embodiments, the crosslinkers may include chemical agents like tripolyphosphate, genipin, bases like NaOH or ammonia salts, calcium salts; or other polymers like sodium alginate, cellulosic polymers like hydroxyl -propylmethyl cellulose (HPMC), Na-CMC, PVP, acrylic polymers like carbopol.

[044] In embodiments, the barrier film contains a first non-alginate polymer and a second non alginate polymer. The second non-alginate polymer can be added to help improve the mucoadhesive properties of the resultant barrier film. Additional mucoadhesive polymers like xanthan gum, cellulose, poly(acrylate), hyaluronon, amberlite resin, or gellan gum may be included to form a firmer bioadhesive film. Specifically a carbopol like polycarbophil or hydroxyl-propylmethyl cellulose (HPMC) can be added as a second non-alginate polymer. In embodiments, the non-alginate polymer is in a range from 1 to 6 % by weight of the total weight of barrier film.

[045] In embodiments, the barrier film includes a microbicidal agent. The microbicidal agent includes one or more of metal nanoparticles or alloy nanoparticles. Alternatively, the barrier film includes microbicidal agent with one or more of benzalkonium chloride or other viricidals. In some embodiments, the barrier film can have a combination of nanoparticles and viricidals.

[046] In embodiments, the microbicidal agents could be chitosan polymer or modified chitosans, metal particles like silver, copper, zinc, antimicrobial agents like benzalkonium chloride (BKC), cetrimide, benzethonium chloride, polyhexamethylenebiguanide (PHMB), xibornol, cresylic acid, antibiotics like Neomycin, Framycetin, Silver Sulphadiazine, Cephalexin,. In some embodiments, the metal particles can be in the form of nanoparticles. For example, metal particles with sizes in the range of 10-500 nm can be used. In some embodiments, micron sized metal particles can be used. Microbicidal could also be herbal extracts / compounds, such as, curcumin, catechins, extract of ginger, garlic, green tea, and such other herbs known to have microbicidal properties.

[047] In embodiments, the microbicidal agents can be dispersed in the film. For example, dispersants and stabilizers can be used to disperse the nanoparticles. The microbicidal agent like benzalkonium chloride, nano-copper, or antibacterial agents/antibiotics like, ampicillin, bacitracin, clindamycin can be dispersed in the polymer to form in the barrier film. In some embodiments, the nanoparticles can be covalently bonded to the polymers. For example, the metal nanoparticles can be covalently bonded with the polymer. In embodiments, the metal nanoparticles can be covalently linked to the non-alginate polymer. In embodiments, the metal nanoparticles can also be linked using coordination bonds with non-alginate polymer.

[048] In embodiments the microbicidal could be pathogen-specific or broad-spectrum IgG derived from animal or avian sources covalently bound to functionalized chitosan. These molecules can be obtained by hyper-immunizing cows or mammalian domestic animals or birds against specific pathogens and the specific IgG from colostrums or milk secreted by these cows can be purified and attached to functionalized chitosan.

[049] In embodiments, the functionalized chitosan, thiolated chitosan or PEGylated chitosan can be bonded to the metal nanoparticles to prevent leaching of the nanoparticles from the barrier film.

[050] In embodiments, the barrier film includes a cros si inked alginate polymer. The alginate polymer is cross-linked by one or more of calcium or magnesium. In embodiments, the alginate polymer includes sodium alginate. In embodiments, the alginate polymer is in a range of 0.5 to 4% by weight of the barrier film.

[051] In embodiments, the barrier film includes one or more stabilizing agents. For example, the stabilizing agents can include buffers to maintain the pH and prevent any type of irritation of the particular biological tissue concerned like skin, wound surface, nasal mucosa, uterine mucosa or the teat canal. In embodiments, the stabilizing agents can include antioxidants and chelating agents like metabisulphite, EDTA salts to prevent oxidation of labile compounds. In embodiments, the stabilizing agents can also include osmotic agents including electrolytes or carbohydrates like mannitol, NaCl, sorbitol, dextrose to maintain the tonicity; antimicrobial preservatives like BKC, benzethonium chloride, parabens, benzoic acid, sorbic acid. In embodiments, the stabilizing agents are added to ensure that formulation and barrier film produced will be stable and safe in terms of being non-irritant and non-sensitizing to the biological tissue like the skin surface, teat canal, uterine mucosa or nasal mucosa.

[052] In embodiments, the barrier film is a hydrogel. For example, the hydrogel contains crossl inked polymer with substantial amount of water. The water helps in keeping the biological tissue hydrated while also protecting it against pathogens. The water will be dispersed following a process so as to release the same over a period of time. In embodiments, the barrier film includes a gel with a residence time of at least 6 hours in/on the mucosal surface if not manually or forcefully expelled. [053] In embodiments, the barrier film has a porosity of less than 0.2 microns. This helps in excluding bacteria just by size exclusion. For example, the bacteria cannot pass through the barrier film and thus will not be able to get through the biological tissue. In embodiments, the barrier film acts as a physical barrier to pathogens like bacteria. In embodiments, the barrier film contains microbicidal agents that attack the bacteria, viruses and other microbes to prevent invasion into the biological tissue. In embodiments, the barrier film is flexible. For example, the barrier film has an elongation to break of at least 50% or higher. In some embodiments, the barrier film can be folded onto itself without cracking. In embodiments, the barrier film can be flexed when applied to skin without substantial cracking. In case of pet animals, the barrier film may be rendered lick-proof. (For birds the application could be through vaporization to enable mass treatment).

[054] In embodiments, the biological surface 14 includes a nasal surface, skin, wound surface, a uterine surface, a vaginal surface, a cervical surface, an ocular surface, an oral surface, or a surface of an animal teat. For example, the biological surface can be part of the human body or animal body.

[055] FIG. 2 illustrates a kit with a first and a second container. The system 20 contains a kit 22 with a first container 24 and a second container 26. In embodiments, the first container 24 contains a composition A and the second container includes a composition B. In embodiments, the composition A includes a non-alginate polymer, a crosslinker, and a microbicidal agent. For example, composition A can contain Chitosan(0.25-2 % w/v) or thiolated or PEG grafted Chitosan and Calcium Chloride (1% w/v) along with a humectant like Glycerol or Propylene Glycol or Sorbitol. In embodiments, the composition B includes a sodium alginate.In some embodiments, the composition B also includes excipients. For example, composition B can be composed of crosslinking polymers including sodium alginate (1-4% w/v) and/or HPMC/ Na- CMC (0.5-2% w/v), Carbopol 934 (0.1-2%w/v). In embodiments, composition A and composition B when mixed create a gel through a sol-gel transformation (in-situ gelling) on contact with the biological surface to form the barrier film. For example, the gel formed acts as a barrier film to stop pathogens from invading the biological tissue. In embodiments, one or both of composition A and composition B may contain additives to stabilize and preserve the formulation and the formed barrier film. [056] In embodiments, the first container and second container do not share a wall and are distinctly separate from each other. In other embodiments, the first container and the second container are secured to each other. In embodiments, the first container may include a device. In embodiments, the second container may include another device. For example, the device can be a spray device, a dropper device, a dipping device or a vaporization device. In embodiments, first container and second container can be part of a single device. For example, first container can be a spray device and second container can also be a spray device. In some embodiments, first container and second container can be different devices. For example, first container may be a spray device and the second container may be a dipping device. In embodiments, the spray device includes a spray nozzle that dispenses the composition and an actuator that triggers the dispensing of the composition. For example, the dispenser and actuator are connected to the enclosure that holds the composition. In embodiments, the dropper device can dispense drops of composition on to the biological tissue. In embodiments, the biological tissue can be dipped into a dipping device to create a thin film of the composition on the surface.

[057] In embodiments, the barrier film 12 from FIG.l is prepared by combining a composition A and a composition B to form the gel. For example, the composition A contains the non- alginate polymer, a crosslinker, and the microbicidal agent; and the composition B contains an alginate polymer. In embodiments, the gel in barrier film 20 is formed by combining composition A and composition B for a period of less than one minute. In embodiments, the barrier film 20, where the composition A contains the non-alginate polymer in a range from 0.5 to 5 % by weight of composition A. In embodiments, the barrier film 20, where the composition B contains the alginate polymer in a range from 1 to 5 % by weight of composition B.

[058] In embodiments, the composition A and composition B are present in a ratio in a range of 1:1 to 1:4 by volume.This will be based on the grades of the chitosan/modified chitosans and the other polymers like alginate, cellulosics, Carbopol® or xanthan gum/gellan gum being included, the pH and environmental factors related to the tissue/biological surface and the crosslinkers used, as well as mechanism of triggering the sol-gel transformation.

In embodiments, the first container and the second container can be made up of plastic, metal, alloy, paper, glass or a combination thereof.

[059] FIG. 3A illustrates a dispensing device for creating a barrier film wherein containers are connected, with the dispensers being on the same side. The dispensing device 30 is a dispensing device for forming a barrier film. The device contains a first enclosure 36 defining a first volume 40. The first enclosure includes composition A. The composition A contains a non-alginate polymer, a crosslinker, and a microbicidal agent. The dispensing device 30 contains a second enclosure 46 defining a second volume 50 comprising a composition B, wherein the composition B contains a sodium alginate. The first dispenser 32 is coupled to the first enclosure 36. The second dispenser 42is coupled to the second enclosure 46. The first conduit 36 fluidically couples the interior of the container or the first volume 40 to the dispenser 32. For example, if the container 40 includes a liquid, it can be transported through the conduit 38 to the dispenser 32 so that it is applied to the biological surface. Similarly, the second conduit 48 fluidically couples the interior of the container or the second volume 50 to the dispenser 42. For example, if the container 50 includes a liquid, it can be transported through the conduit 48 to the dispenser 42 so that it is applied to the biological surface. In embodiments, the first actuator 34 is coupled to the first dispenser 32 and the second actuator 44 is coupled to a second dispenser 42. In embodiments, the first actuator 34 is configured to cause composition A to be released from the first dispenser 32 and the second actuator 44 is configured to cause composition B to be released from the second dispenser 42.

[060] In embodiments, the dispensing device 30 has the first actuator 34 and the second actuator 44connected to a single actuator. For example the single actuator can be configured to release composition A from the first dispenser 32 and composition B from second dispenser 42 at specified intervals of time. For example, the single actuator is configured such that second dispenser 42 dispenses composition B ten seconds after first dispenser 32 dispenses composition A. In embodiments, the single actuator is configured such that the second dispenser dispenses composition B immediately after the first dispenser dispenses composition A.

[061] In embodiments, the dispensing device 30 is configured to release composition A and composition B directly on the biological surface. In embodiments, the dispensing device is designed such that the first enclosure and second enclosure are pressurized. In some embodiments, the dispensing device is designed such that the first enclosure and the second enclosure are at atmospheric pressure.

[062] In embodiments, the dispensing device consists of one or more of glass, ceramic, polymer, paper, metal, or alloy. In embodiments, the enclosure can be made up of plastic, metal or glass. For example, plastics may be high density polyethylene (HDPE), or polystyrene, or polypropylene; glass if used should be Type I to ensure no leaching of alkali. Metal containers may be stainless steel to ensure freedom from corrosion; or aluminum or tinplate containers when used should be suitably lacquered with resins/polymer coatings. Suitable rubber components, if present as washers should be free from leachables. In embodiments, the paper can be a single layer or can be coated with polymer to help make it usable. In some embodiments, the paper may be a composite construction laminated with other polymer.

[063] In embodiments, the dispensing device consists of comprising a power source for powering the actuator. For example, the dispensing device may be powered by a portable battery or a rechargeable battery.

[064] In embodiments, one or both of the first dispenser or the second dispenser includes a spray nozzle, a dropper, a brush, or a foam applicator. For example, the first dispenser can be a spray nozzle and the second disperser can also be a spray nozzle. In other examples, the first dispenser can be a dropper and the second dispenser can be a dropper. In embodiments, the first dispenser and the second dispenser are not the same type. For example, the first dispenser can be a spray nozzle and the second dispenser can be a dropper.

[065] FIG. 3B. illustrates a dispensing device for creating a barrier film wherein containers are connected, with the dispensers being on opposite sides. The dispensing device 50 is a dispensing device for forming a barrier film. The device contains a first enclosure 56 defining a first volume. The first enclosure includes composition A. The composition A contains a non-alginate polymer, a crosslinker, and a microbicidal agent. The dispensing device 50 contains a second enclosure 64 defining a second volume comprising a composition B, wherein the composition B contains a sodium alginate. The first dispenser 52 is coupled to the first enclosure 56. The second dispenser 68 is coupled to the second enclosure 64. The first conduit 58 fluidically couples the interior of the container or the first volume to the dispenser 52. For example, if the container 56 includes a liquid, it can be transported through the conduit 58 to the dispenser 52 so that it is applied to the biological surface. Similarly, the second conduit 62 fluidically couples the interior of the container or the second volume to the dispenser 68. For example, if the container 64 includes a liquid, it can be transported through the conduit 62 to the dispenser 68 so that it is applied to the biological surface. In embodiments, the first actuator 54 is coupled to the first dispenser 52 and the second actuator 66 is coupled to a second dispenser 68. In embodiments, the first actuator 54 is configured to cause composition A to be released from the first dispenser 52 and the second actuator 66 is configured to cause composition B to be released from the second dispenser 68.

[066] FIG. 4A illustrates a method of dispensing from the dispensing device wherein the composition A and composition B are added one after the other. In embodiments, the system 60 contains the dispensing device 62 dispenses composition A on the biological surface 68. The dispensing device 64 dispenses composition B on the biological surface after composition A. For example, composition A is coated on the biological surface 68 after composition B to create a barrier film 66. In other examples, composition B is coated on the biological surface after composition A to create a barrier film 66.

[067] FIG. 4B. illustrates a method of dispensing from the dispensing device wherein the composition A and composition B are dispensed so that both reach the barrier film at the same time. In embodiments, In embodiments, the system 70 contains the dispensing device 72 dispenses composition A on the biological surface 78. The dispensing device 74 dispenses composition B. In embodiments, the device is configured in way to dispense composition A and composition B around the same area at the same time. For example, the composition A and composition B reaches the biological surface 78 at the same time to create a barrier film 76.

For example, composition A and composition B do not substantially mix with each other until it reaches the biological surface 78.

[068] FIG. 5 illustrates a block diagram of method of formation of barrier film. In embodiments, the present disclosure describes a method for forming a barrier film 80 against pathogens. The barrier film includes a gel. The method includes applying composition A (82) on a biological surface, wherein the composition A includes a non-alginate polymer, a crosslinker, and a microbicidal agent. Further, the method includes applying a composition B (84) on the biological surface, wherein the composition B includes a sodium alginate. The gel is formed on the biological surface by combination of the composition A and the composition B (86). In embodiments, composition A is applied on the biological surface followed by composition B is applied to the biological surface. In embodiments, composition B is applied on the biological surface followed by composition A is applied to the biological surface. In embodiments, composition A and composition B are applied at the same time on the biological surface.

[069] In embodiments, the present disclosure describes a method for forming a nasal barrier film. The method involves spraying a composition A on the nasal mucosal surface, wherein the composition includes a mucoadhesive polymer, which can be chitosan or modified chitosan, such as, thiolated chitosan along with a crosslinker, and a microbicidal agent like and spraying a composition B over composition A, wherein the composition B includes a sodium alginate, , excipients to create sol gel transformation {in situ gelling) on contact with the mucosal surface to form the barrier film, as well as additives to stabilize and preserve the formulation and the formed barrier film. This allows the composition A and composition B to crosslink and form a sol-gel film by crosslinking and phase transformation on the nasal mucosal surface. This phase transformation can be triggered by factors like temperature, pH of nasal secretions or presence of lysozyme or ions in nasal secretions.

[070] Another application of the barrier film could be wound dressing spray where the film will keep the abraded surface insulated from entry of external pathogens, keep the wound hydrated and hence expedite wound healing. A chitosan reinforced alginate film will be formed after spraying Solution A containing chitosan and calcium and Solution B consisting of alginate and excipients will lead to formation of a barrier film impermeable to bacteria.

[071] In embodiments, the present disclosure describes a method of forming a wound barrier film. The method involves spraying composition B over composition A. Composition A contains a non-alginate polymer, by adding polymer, which can be gellan gum, and in addition may include a microbicidal agent, including Ag nano-systems or PHMB or benzalkonium chloride, and spraying a composition B over composition A, wherein the composition B includes a sodium alginate, excipients to create sol gel transformation {in situ gelling) on contact with the wound fluids to form the barrier film, as well as additives to stabilize and preserve the formulation and the formed barrier film. In embodiments, composition A is sprayed over composition B.

[072] In embodiments teat spray / dip which consisted of thiolated chitosan capped with silver nanoparticles in solution A was used whereas solution B consisted of sodium alginate.

[073] In embodiments, the present disclosure describes a method of manufacturing a barrier type teat spray or a dip composition for bovine, buffaloes and ovine. The method involves creating composition A by adding chitosan or modified chitosan, along with a crosslinker, and a microbicidal agent and spraying or a dip composition B over composition the A, wherein the composition B includes a sodium alginate, excipients to create sol gel transformation {in situ gelling) on contact with the teat surface to form the barrier film, as well as additives to stabilize and preserve the formulation and the formed barrier film. This barrier film is particularly useful for postpartum treatment in animals, particularly bovine animals. The method involves creating composition A by adding a chitosan or modified chitosan, along with a crosslinker, and a microbicidal agent like Ag in nanoform, which may be cross-linked to polymer surface and creating composition B by adding sodium alginate excipients to create sol gel transformation {in situ gelling) on contact with the uterine mucosal surface to form the barrier film, as well as additives to stabilize and preserve the formulation and the formed barrier film. In some embodiments, the microbicidal is not required as the pore size is less than 0.2 microns, that does not let bacteria to enter due to size exclusion.

[074] In embodiments, the present disclosure describes a method of manufacturing of the spray formulation compositions. In embodiments, composition A composed of a polymer which is bioadhesive, and may be included from polymers like gellan gum, chitosan or modified chitosan derivatives like thiolated or PEGylated Chitosan with enhanced adhesiveness to biological tissues, alongwith a crosslinker, and a microbicidal agent. In embodiments, the composition B includes a sodium alginate, and excipients to create sol gel transformation {in situ gelling) on contact with the biological surface to form the barrier film, as well as additives to stabilize and preserve the formulation and the formed barrier film. In embodiments, manufacturing of compositions A and B may be done separately and will involve the following steps- hydration of the bioadhesive polymer by soaking in 50% of the solvent or humectant like glycerol, to allow complete swelling of the polymer, period of soaking ranging from 3 hours to 24 hours, and can be accompanied by stirring at RT. The crosslinker and microbicidal agent and any other additives can be dissolved in remaining solvent, followed by mixing with the polymer solution under mechanical stirring. This is followed by filling of the two compositions in the spray kit in the respective compartments, stoppering and sterilization by autoclaving. Other alternative - the two compositions are prepared in jacketed vessels under pressure (simultaneous sterilization) followed by filling in sterile spray device.

EXAMPLES

Unless otherwise noted, all parts, percentages, ratios, etc. in the examples and the rest of the specification are by weight. TABLE 1 lists materials used in the examples and their sources.

TABLE 1

Example 1

[075] Standardization of reagents: In order to remain intact in situ for the desired time it is necessary to standardize the concentrations of alginate in solution A as this concentration will decide the thickness, strength and adherence of the film to the tissues. A teat dip product for use in bovine was developed. The teat dip consisted of solution A containing chitosan and calcium and solution B consisting of alginate and glycerol. [076] Preparation of Solution A: In order to optimize concentration, 0.25 to 1.5 g chitosan was dissolved in 100 ml 1% (v/v) acetic acid solution with constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v), methyl paraben 0.5% and propyl paraben 0.05% were added. Solution 2 was prepared by dissolving 2.5 g sodium alginate in 100 ml distilled water heated at 90°C with stirring and glycerol 1% v/v was added with stirring for a period of 10 minutes. Preservatives methyl paraben (0.5%) and propyl paraben (0.05%) are added to the solution. The two solutions are sterilized at 121°C and 15 psi for 15 minutes. For in vitro studies the barrier film was formed on circular Whatman No. 1 filter paper. Solution A was sprayed first followed by spray B and the film was allowed to firm up for 5 minutes. The viscosity was measured by Brookefield Viscometer (Model LVT) spindle No. 34 at 25°C and thickness of the film was studied using vernier caliper. TABLE 2 describes the results of this experiment. Since 1% chitosan yielded the desired type of film this concentration was used in further teat dip studies.

[077] The standard concentration of chitosan could vary depending upon the tissue on which the barrier film is desired and the desired film residency period on the tissue.

TABLE 2 TABLE 3

[078] Preparation of Solution B: In order to optimize the concentration of sodium alginate 0.5 to 2.5 g was dissolved in distilled water heated at 90°C with stirring and glycerol 1% v/v is added with stirring for a period of 10 minutes. Solution A was prepared by dissolving 1 g chitosan in 100 ml 1% (v/v) acetic acid with constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v) and preservatives, methyl paraben 0.5% and propyl paraben 0.05% were added. The two solutions are sterilized at 121°C and 15 psi for 15 minutes. For in vitro studies the barrier film was formed on circular Whatman No. 1 filter paper. Solution A was sprayed first followed by spray B and the film was allowed to firm up for 5 minutes. Time required for film formation was noted. Properties of the film such as thickness, viscosity, folding endurance were studied using standard methods. TABLE 3 shows the results of the experiment on standardization of sodium alginate concentration. Based on the results sodium alginate concentration was fixed at 2.5% for further studies on teat dipping spray.

[079] Studies to prove barrier properties: The principle of the test is that if the film is a physical barrier to pathogenic bacteria, in the invitrosystem it would not permit permeation of bacteria from the film onto nutrient agar gel. The study was done for both types of films, the one formed after spray and the other when it is formed after dipping. Spraying or dipping of sterile water was used as control.

[080] Work flow of barrier study: To simulate bovine mammary teats, the finger part of the rubber gloves or the rubber teats was first nicked open in the bottom so as to mimic the teat opening and sphincter. The separated rubber teats, in triplicate, were filled with nutrient agar and allowed to solidify. The rubber teat was dipped in solution A and then Solution B and allowed to dry to form the barrier film. The rubber teats were then kept dipped in ‘Tarson’ tube containing sterile sand mixed with staphylococcus or E.coli culture and little water to mimic contaminated surface of the barn floor. After 24 hours the rubber teat was taken out of the Tarson tube and the agar stub streaked onto a solid agar in a petri dish. The petri dish was incubated at 37 °C for 24 hours. In the dipping method instead of spray the glove finger containing solidified nutrient agar was dipped in solution A followed by solution B for 15 seconds each and placed in the Tarson tube containing Staphylococcus aureus or E.coli for 12-24 hours. The rubber teat was then streaked on to solidified nutrient agar in a petri dish and incubated for a period of 24 hours to observer bacterial growth. FIG. 6 shows the barrier film formed on the rubber teat after dipping in solution A and solution B.

[081] FIG. 7 is a photograph showing microbial plates comparing positive and negative controls with three samples -ve: negative control, +ve: positive control. The positive control rubber teat was not dipped / sprayed with the formulation, only agar stick dipped in sand and microbial culture; Negative Control: No formulation, No culture, only agar stick.; Sample: Solidified agar stick dipped into the formulation. It was then dipped into sand and microbial culture. After incubation of the streaked samples onto sterile nutrient agar plates, no growth was seen with any of the samples (FIG.7) confirming that there was no permeation of bacteria from the sand to the agar stub. The control test however, showed growth of the bacteria. This proves the barrier property of the film. Since it is known that after milking is over the teat sphincter in animals remain relaxed for a period of around 2-3 hours hence the film must remain intact during the period. Since the animal would remain active in the barn and may sit on the floor several times, the barrier film should be resilient to breaking due to shear pressure. This property can be studied in vitro and is termed ‘Folding endurance test’. This is estimated by double folding the film at the same point repeatedly. The number of folds after which the film breaks are termed ‘Folding endurance’. The results show that the film did not break even after 322 times double fold, giving a fold endurance value of 2.509 which is considered as high to withstand wear and tear.

[082] Example 2: The rubber teats dipped in normal disinfectant did not prevent entry of bacteria. This study followed the protocol described in Example 1 except that instead of dipping the rubber teats in the solution A and Solution B, it is dipped twice in PHMB disinfectant solution. The result after exposing the rubber teats for 24 hours in bacteria-contaminated sand showed growth of the bacteria in the nutrient agar. This result proved that only dipping in a disinfectant solution is not enough to prevent entry of bacteria from the teat sphincter.

[083] Example 3: Spraying of chitosan alone or sodium alginate alone did not result in barrier film formation. In this study the experimental protocol was similar to described in Example 1 except that instead of dipping in both solution A and solution B, the rubber teats were dipped in either of the solutions. After exposing the agar filled rubber teats to the bacteria- contaminated sand for 24 hours, the exposed agar stub streaked on the nutrient agar in petri dishes showed extensive growth of the colonies proving that use of only single polymer solution did not result in forming of an effective barrier film.

[084] Example 4: Thiolated-chitosan-silver-nanoparticle-alginate Barrier type Teat Dip.

Thiolated chitosan is obtained commercially. Silver nano-particles is prepared using sodium citrate as the reducing agent as per a method developed in the inventors’ laboratory. Capping of thiolated chitosan with silver nanoparticles is done as per standard published method. Solution A is prepared by dissolving lg thiolated-silver nanoparticle-bound-chi tosan in 100 ml 1% (v/v) acetic acid solution with constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v), methyl paraben 0.5% and propyl paraben 0.05% is added. Solution B is prepared by dissolving 2.5 g sodium alginate in 100 ml distilled water heated at 90°C with stirring and glycerol 1% v/v, gellan gum 0.4-0.6% w/v and PEG is added with stirring for a period of 10 minutes. Preservatives methyl paraben (0.5%) and propyl paraben (0.05%) are added to the solution. Spray solutions A and B are sterilized by autoclaving at 121°C and 15 psi for 15 mins. The solutions are then filled into sterile conjoined spray bottles A and B, respectively and sealed with screw capped nozzles. The barrier-property of the film in the in-vitro rubber tube model described in Example 1 is studied. The rubber teats were dipped in sand contaminated with 10- times load of the S. aureus or E. coli than the method described in Example 1. The results show that the barrier film does not allow permeation of bacteria into the rubber teat and acts as perfected barrier at the teat opening.

[085] Example 5: Barrier Type Wound Spray. Solution A was prepared by dissolving lg chitosan in 100 ml 1% (v/v) acetic acid solution prepared by constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v), methyl paraben 0.5% and propyl paraben 0.05% were added. Solution 2 is prepared by dissolving 2.5 g sodium alginate in 100 ml distilled water heated at 90°C with stirring and glycerol 1% v/v is added with stirring for a period of 10 minutes. Preservatives methyl paraben (0.5%) and propyl paraben (0.05%) are added to the solution.

[086] Spray solutions A and B were sterilized by autoclaving at 121°C and 15 psi for 15 mins. The solutions were then filled into sterile conjoined bottles A and B of the spray kit, respectively and sealed with screw capped nozzles.

[087] In order to treat an open surgical or traumatic wound, the wound was first decontaminated with commercially available wound dressing disinfectant. The pilot study was carried out in animals suffering from traumatic wound in selected dairy farms under the supervision of the local veterinarian and permission of the owner of the animal. Solution A was sprayed over the wound surface followed by Solution B, when formation of an in situ barrier film is triggered in contact with wound fluids and the film was formed rapidly in 2-3 minutes. The wound is then left undressed. Depending upon the location and size of the wound the spray treatment is carried out daily or alternate days for a period of 5-7 days. The wound for healing is examined after 5 days of the first treatment. Wound healing is indicated by complete covering of the wound with granulated tissues and appearance of normal skin on the wound surface.

[088] Example 6: Barrier film is prepared using the method outlined in Example 5 except silver nitrate nano-particles-capped thiolated chitosan is used in solution A, instead of chitosan. Studies to prove immobilization and minimum release of silver nano-particles is carried out as per standard methods. The folding strength of the barrier film is carried out as per the method described in Example 1. The efficacy studies is carried out in selected traumatic wound cases in domestic animals following the ethical practice of owners’ permission and supervision of the local veterinarian in charge of the animals’ treatment.

[089] Example 7: Barrier film is prepared using the method outlined in Example 5 except instead of silver nanoparticles, other disinfectant, such as, organic iodine, PHMB or benzalkonium chloride is added in solution A consisting of deacetylated chitosan. The efficacy study is carried out in traumatic wound as described in Example 6.

[090] Example 8: Barrier film is prepared using the method outline in Example 5 except solution A contains dispersed antibiotics or antibacterial, such as, polymyxin B, neomycin, gentamycin, bacitracin, mupirocin, etc., instead of silver nanoparticles. These antibiotics are released in therapeutic doses over a time period. The efficacy study is carried out in traumatic wound as described in Example 6.

[091] Example 9: Barrier film with natural microbicidal. Barrier film is prepared using the method outlined in Example 5 except solution A contains a natural microbicidal substance such as, curcumin, mixture of extracts of herbs known to have additional healing properties instead of silver nanoparticles. The efficacy study is carried out in traumatic wound as described in Example 6.

[092] Example 10: Barrier Type Nasal Spray. Solution A is prepared by dissolving lg thiolated-silver nanoparticle -bound chitosan in 100 ml 1% (v/v) acetic acid solution with constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v), methyl paraben 0.5% and propyl paraben 0.05% is added.

[093] Solution B is prepared by dissolving 2.5 g sodium alginate in 100 ml distilled water heated at 90°C with stirring and glycerol 1% v/v is added with stirring for a period of 10 minutes. Preservatives methyl paraben (0.5%) and propyl paraben (0.05%) are added to the solution. The two solutions are sterilized at 121°C and 15 psi for 15 minutes. The prepared solutions are evaluated for clarity, pH, rheology, zeta potential, viscosity, contact angle, surface tension, droplet size distribution, muco-adhesiveness, barrier and microbicidal property and stability. The formulation is also assessed for their effects on nasal epithelial cells. The study to show barrier property to virus entry is performed in 12-14 days embryonated using New Castle Disease Virus as the representative of the encapsulated virus family. A small size perforation is drilled on air sac side of the egg and sealed with an adhesive dipped in solution A and solution B. A drop of virus is smeared in the sealant and allowed to form a gel. The eggs are then re-incubated in the inverted side so that the embryonal fluid will remain in contact with the sealant membrane. The eggs are candled every alternate day to examine embryo survival. In negative control no spray is used on the sealant adhesive film. The persistency of the barrier film is also studied in goat nasal model obtained from the slaughterhouse. [094] The formulation exhibits suitable viscosity and rheological properties. Spray droplets are in ranges of 10-40 pm, which is suitable for nasal administration. The adhesion of the formulation isin the range of to enable formation of a film 8-4.0 and 0.2-27 pj, respectively. In addition, the formulation shows no signs of cytotoxicity and does not open the tight junction of nasal epithelial cells. The egg embryo study results show that the barrier film does not permit entry of virus in the embryonal eggs. The study on goat nasal tissue model shows that the film remains persistent for 6-8 hours.

[095] Example 11: Chitosan-sodium alginate barrier film. Solution A is prepared as described in Example 10 except the desired microbicidal is dispersed with silver in solution A in place of thiolated chitosan. Similar studies as described in Example 10 are performed. The results show that the barrier film remained in situ for more than 6 hours. In vitro release studies show that silver nanoparticles are released for a period of 8 hours.

[096] Example 12: Thiolated Chitosan-alginate barrier film for cervico -uterine Infusion.

This formulation is made in the form of infusion rather than the spray, but the basic principle of the product remains the same. Solution A is prepared by dissolving lg thiolated- silver nanoparticle -bound chitosan in 100 ml 1% (v/v) acetic acid solution with constant stirring @600 rpm for 15 minutes. To this calcium chloride 1% (w/v), methyl paraben 0.5% and propyl paraben 0.05% are added. Solution B is prepared by dissolving 2.5 g sodium alginate in 100 ml distilled water heated at 90°C with stirring and glycerol 1 % v/v is added with stirring for a period of 10 minutes. Preservatives, methyl paraben (0.5%) and propyl paraben (0.05%) are added to the solution.

[097] Spray solutions A and B are sterilized by autoclaving at 121°C and 15 psi for 15 mins. The solutions are then filled into sterile conjoined spray bottles A and B, respectively and sealed in a 50 ml screw-capped glass vials. For in vitro study cervico-uterus part is dissected from freshly slaughtered buffalo. The administration is intrauterine deposition of the solution A in cervico-uterine area followed by deposition of solution B. While withdrawing the uterine infusion gun 10 ml of each solution is also deposited in the mid cervix to form a barrier seal. The organ is kept immersed in a tank containing glucose phosphate buffer. After 8 hours the uterus and cervix is dissected to study persistency of the film and the seal. The results show that there is a uniform barrier film coating the uterine mucous membrane and a seal in the cervix. [098] Example 13: Chitosan- alginate barrier formulation for cervico-uterine Infusion.

The formulation is developed for situations where release of microbicidal is desired over a period of time to take care of constant stream of infection. The formulation is similar to the one described in Example 12 except that instead of thiolated chitosan, deacylated chitosan was used and the silver nano-particles is dispersed in chitosan using standard dispersal technique. The method of evaluation of the barrier film property was done in similar manner as described in Example 12.

[099] The details of one or more examples are set forth in the accompanying drawings and description. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

[0100] Various aspects of the invention have been described. These and other aspects are within the scope of the following claims.

[0101] Aspect A. A barrier film against pathogens, the barrier film comprising: a gel comprising a non-alginate polymer and a cross-linked alginate polymer; and a microbicidal agent in the gel. [0102] Aspect B. The barrier film of aspect A, wherein the non-alginate polymer comprises a chitosan polymer.

[0103] Aspect C. The barrier film as in one of aspects A or B, wherein the non-alginate polymer consists of the chitosan polymer.

[0104] Aspect D. The barrier film as in one of aspects A-C, wherein the chitosan polymer is a non-functionalized chitosan, a thiolated chitosan or a PEGylated chitosan.

[0105] Aspect E. The barrier film as in one of aspects A-D, wherein the chitosan polymer is crossi inked with trio-poly-phosphate.

[0106] Aspect F. The barrier film as in one of aspects A-E, wherein the non-alginate polymer is a first non-alginate polymer, wherein the gel comprises a second non-alginate polymer.

[0107] Aspect G. The barrier film of aspect F, wherein the second non-alginate polymer comprises one or more of cellulose, poly( acrylate), hyaluronon, amberlite resin, or gellan gum. [0108] Aspect H. The barrier film as in one of aspects A-G, wherein the microbicidal agent is covalently linked to the non-alginate polymer.

[0109] Aspect I. The barrier film as in one of aspects A-H, wherein the microbicidal agent comprises one or more of metal nanoparticles. [0110] Aspect J. The barrier film as in one of aspect A-I, wherein the microbicidal agent comprises one or more of benzalkonium chloride.

[0111] Aspect K. The barrier film as in one of aspect A-J, wherein the crosslinked alginate polymer is cross-linked by one or more of calcium or magnesium.

[0112] Aspect L. The barrier film as in one of aspect A-K, wherein thenon-alginate polymer is in a range from 1 to 6% by weight.

[0113] Aspect M. The barrier film as in one of aspect A-L, wherein the alginate polymer comprises sodium alginate.

[0114] Aspect N. The barrier film as in one of aspect A-M, wherein the alginate polymer in a range of 0.5 to 4% by weight.

[0115] Aspect O. The barrier film as in one of aspect A-N, wherein the barrier film further comprises one or more stabilizing agents.

[0116] Aspect P. The barrier film as in one of aspect A-O, wherein the barrier film comprises a hydrogel.

[0117] Aspect Q. The barrier film as in one of aspect A-P, wherein the gel has a shelf-life of at least 6 hours if not manually or forcefully expelled.

[0118] Aspect R. The barrier film as in one of aspect A-Q, wherein the gel has a porosity of less than 0.5 microns.

[0119] Aspect S. The barrier film as in one of aspect A-R, wherein the barrier film is flexible. [0120] Aspect T. The barrier film as in one of aspect A-S, wherein the barrier film is prepared by a method comprising combining a composition A and a composition B to form the gel, wherein the composition A comprises: the non-alginate polymer, a crosslinker, and the microbicidal agent; and wherein the composition B comprises an alginate polymer.

[0121] Aspect U. The barrier film of aspect T, wherein the gel is formed by combining composition A and composition B for a period of less than one minute.

[0122] Aspect V. The barrier film as in one of aspects T or U, wherein composition A comprises the non-alginate polymer in a range from 0.5 to 5 % by weight of composition A. [0123] Aspect W. The barrier film as in one of aspects T-V, wherein composition B comprises the alginate polymer in a range of 1 to 5 % by weight of composition B.

[0124] Aspect X. The barrier film as in one of aspects T-W , wherein the composition A and composition B are present in a ratio in a range of 1: 1 to 1:4 by volume. [0125] Aspect Y. A method for forming a barrier filmcomprising a gel against pathogens, the method comprising: applying composition A on a biological surface, wherein the composition A comprises: a non-alginate polymer, a crosslinker, and a microbicidal agent; applying a composition B on the biological surface, wherein the composition B comprisesa sodium alginate; and allowing the gel to form on the biological surface by combination ofthe composition A and the composition B.

[0126] Aspect Z. The method of aspect Y, wherein one or both of the applying of composition A or the applying of composition B comprises one or more ofspraying, dipping, rolling, brushing or spreading.

[0127] Aspect AA. The method as in one of aspects Y or Z, wherein the composition B is applied on the biological surface after applying composition A on the biological surface.

[0128] Aspect AB. The method as in one of aspects Y-AA, wherein the composition A is applied on the biological surface after applying composition B on the biological surface.

[0129] Aspect AC. The method as in one of aspects Y-AB, wherein the composition A is applied on the biological surface after applying composition B on the biological surface.

[0130] Aspect AD. The method as in one of aspects Y-AC, wherein the composition A is applied on the biological surface simultaneously as composition B on the biological surface.

[0131] Aspect AE. The method as in one of aspects Y-AD, wherein the composition A is not combined with composition A before applying on the biological surface.

[0132] Aspect AF. The method as in one of aspects Y-AE, wherein the biological surface comprises a nasal surface, skin, wound surface, a uterine surface, a vaginal surface, a cervical surface, aocular surface, an oral surface, or a surface of an animal teat.

[0133] Aspect AG. A kit comprising a first container and a second container, wherein the first container comprises a composition A, wherein the composition A comprises: a non-alginate polymer, a crosslinker, and a microbicidal agent; wherein the second container comprises a composition B, wherein the composition B comprises a sodium alginate.

[0134] Aspect AH. The kit of aspect AG, wherein the first container and the second container comprises a spray device, a dropper device, a dipping device or a combination thereof.

[0135] Aspect AI. The kit as in one of aspects AG or AH, wherein the first container and second container are secured to each other. [0136] Aspect AJ. A dispensing device for forming a barrier film, the device comprising: a first enclosure defining a first volume comprising a composition A, wherein the composition A comprises: a non-alginate polymer, a crosslinker, and a microbicidal agent; a second enclosure defining a second volume comprising a composition B, wherein the composition B comprises a sodium alginate; a first dispenser coupled to the first enclosure; a second dispenser coupled to the second enclosure; and a first actuator coupled to the first dispenser and the second actuator coupled to a second dispenser, wherein the first actuator is configured to cause composition A to be released from the first dispenser and the second actuator is configured to cause composition B to be released from the second dispenser.

[0137] Aspect AK. The dispensing device of aspect AJ, further comprising a single actuator connected to the first actuator and second actuator; configured to release composition A and composition B at specified intervals of time.

[0138] Aspect AL. The dispensing device as in one of aspects AJ and AK, configured to release composition A and composition B directly on the biological surface.

[0139] Aspect AM. The dispensing device as in one of aspects AJ-AL, wherein the first enclosure and second enclosure are pressurized.

[0140] Aspect AN. The dispensing device as in one of aspects AJ-AM, wherein the device comprisesone or more of glass, ceramic, polymer, paper, metal, or alloy.

[0141] Aspect AO. The dispensing device as in one of aspects AJ-AN, further comprising a power source for powering the actuator.

[0142] Aspect AP. The dispensing device as in one of aspects AJ-AP, wherein one or both of the first dispense or the second dispenser comprises a spray nozzle, a dropper, a brush, or a foam applicator.