Formulation of a composite nasal spray enabling enhanced surface coverage and prophylaxis of SARS-COV-2

Airborne pathogens pose high risks in terms of both contraction and transmission within the respiratory pathways, in particular the nasal region. Although knowledge of airborne transmission has long been known, there is little in the way of adequate intervention that can protect the individual, or even prevent further spread. This study focuses on a nasal applicant with the capacity to combat such issues, by focussing on the SARS-CoV-2 virus. Formulation of a spray containing polysaccharides known for their mucoadhesive properties was undertaken and characterised for their mechanical, spray patterns and antiviral properties. The ability to engineer key behaviours such as yielding have been shown, through systematic understanding of a composite mixture containing two polymers: gellan and λcarrageenan. Furthermore, spray systems demonstrated highly potent antiviral capacities, resulting in complete inhibition of the virus when studied for both prophylaxis and prevention of spread. Finally, a mechanism has been proposed to explain such findings. Therefore, demonstrating the first fully preventative device, targeted to protect the lining of the upper respiratory pathways.


Introduction 27
Transmission of viruses occurs through 4 routes: direct contact, via physical contact with a 28 carrier; indirect contact, interactions with contaminated objects; droplet and airborne 29 transmission, often through coughs, sneezes and breathing; and, aerosolization, atomised 30 virus suspended in airflow. [1] Airborne transmission of respiratory pathogens, whether 31 through droplets or atomisation, is particularly deleterious, with the virus effectively and 32 locally delivered to the respiratory pathways. Recent work, primarily undertaken within the 33 COVID-19 pandemic has heavily focused on providing a deeper understanding on person to 34 person airborne transmission. [2][3][4][5] During the act of coughing, turbulent air forces mucus 35 breakup into droplets [6] (ca. 0.62 to 15.9 µm), [7] which are then expelled through the oronasal 36 passages at flow rates of up to 12 Ls -1 , reaching velocities of up to 30 ms -1 . [8] Unfortunately, 37 the expelled cloud is subject to many varying parameters: speed of expulsion, droplet size 38 and environmental effects such as air speed, resulting in effected boundaries ranging from ca. 39 0.5 to 8 m. [8,9] It is likely, for this reason, that the inability to standardise transmission in this 40 way has led to an ongoing lack of change in regard to the concepts employed to cope with 41 such issues; [10] with recommended distancing guidelines still based on the ideas portrayed by 42 Chaplin and Wells close to a century ago. [11,12] Although the epidemiology of the severe acute 43 respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet definitive, clear indications 44 suggest epidemiological characteristics closely linked to airborne transmittance [13] . 45 46 There are many airborne viruses including: influenza-, rhino-, adreno-, entero-and corona -47 virus. The latter, coronaviridae (CoVs) family, are implicated in a variety of gastrointestinal, 48 interaction with the mucus; known as mucoadhesion. A range of polymers known for their 98 mucoadhesive properties (gellan, carrageenan, alginate, pectin, dextran) were screened 99 through spray application to a 45° acetate surface (Fig. 1b). Polymers were classed for their 100 ability to create an even coverage whilst being retained at the sprayed site. Fig. 1bii and 1biii  101 shows typical images for several of the polymers tested, demonstrating a "good" and "poor" 102 candidate; gellan and alginate respectively. Screening in this manner provided a means to 103 narrow the systems down to both gellan and carrageenan going forward, with others either 104 creating heterogeneous distributions or flowing under their own mass. 105 106 Flow behaviours were characterised via dynamic viscosity (from high to low shear stress), 107 representative of the material once sprayed. Resultant profiles for the gellan were modelled 108 demonstrating a transition from power law to Cross model, suggesting the loss of a dynamic 109 yield stress to zero-shear viscosity as a function of the polymer concentration (Fig. 1ci) Fig. 1ciii and Table 1. 125 Flow behaviours for the 1% (w/v) systems showed a clear transition from material 126 characteristics indicative of the gellan (viscosity asymptoting at low stresses), to those of the 127 lcarrageenan (plateaued viscosities at low stresses), as the ratio of the two polymers shifted 128 from one extreme to the other (gellan to lcarrageenan). Loss of overall viscosity was also 129 observed as the systems shifted from high to low gellan ratios, confirmed by the reduction in 130 consistency coefficient (K) from 3.54 to 0.03. This correlated well with the increase in rate 131 index (n), where more gellan resulted in higher degrees of shear thinning: 0.40 to 0.82 for 132 100% gellan and 100% lcarrageenan, respectively. A reduction in the total polymer content 133 to 0.4% (w/v) resulted in all mixtures characterised by the Cross model, consistent with data 134 provided for the isolated polymers. Further reduction in the polymer concentration, to 0.2% 135 (w/v), resulted in profiles independent on the ratio of gellan to lcarrageenan, with samples 136 indistinguishable from each other (within error). 137

180
The role that total and ratio of polymer play within the spray-ability of the composite systems 181 can be clearly seen in Fig. 2b. In all instances, irrespective of total polymer concentration, a 182 shift to smaller distributions was observed as the ratio of gellan to lcarrageen decreased. 183 Such changes became more pronounced with total polymer, where the magnitude of change 184 between 100% gellan to 100% lcarrageenan, followed 1.0%> 0.4%> 0.2% (w/v). such 185 observations were mirrored in the total coverage data (Fig. 2cii). Replacing 25% of the total 186 lcarrageenan with gellan resulted in a 4.9% and 4.4% increase in coverage, for the 0.2% and 187 0.4% (w/v) systems; with an initial loss in spray coverage (-3.5%) for the 1% total polymer 188 content. Coverage was further increased to 9.0%, 14.1% and 2.9% for the 0. suppression of the infection, k-, iand l-carrageenan were studied using the SARS-CoV-2 246 assay (Fig. 4c). It was observed that in all cases, where the cells were treated prior to being 247 exposed to the virus, infection was lowered to below the untreated control group (p<0.001). 248 This could not be said for the pre-treated virus, where larger dilution factors (1/1000 and 249 1/3000) did not statistically affect the degree of infection for both the iand l-carrageenans. The role that the nasal passage plays in frontline defence, filtering harmful bacteria and 264 viruses, naturally elevates the sinonasal pathways to high risk, in terms of infection [33] . The 265 need to formulate medicines/devices which can help regulate and protect this area are thus 266 clear, however, like many regions of the body the nasal cavity poses many challenges, due to: 267 ease of access, dynamics (native clearing mechanism) and topology (inclined surfaces or 268 ceilings). As such, formulation engineering plays a decisive role in the design of novel 269 therapeutics. The polymer thus provides a physical role, expanding the hydrodynamic volume around the 361 cell/virus and preventing close proximity. [50] Even though the role that the negatively charged 362 sulphate groups play in the ability to adsorb to the bio-interface, it is unclear from the data 363 whether a link between the degree of sulphation and suppression of infection exists. 364 Although not significant in the role of coating, gellan does demonstrate its applicability when 365 considering prophylaxis through entrapment and elimination. The ability to engineer high 366 viscosities and yielding behaviour at this point becomes key, proportionally slowing 367 diffusivity, as described by the Stokes-Einstein relation. [51] To this end, diffusion of the virus 368 towards the host cells can be hampered within timescales associated with typical nasal 369 clearance. [52] In reality a combination of the 3 proposed mechanisms is likely to occur. To this 370 end, physical entrapment is suggested to provide a first means of defence, simultaneously 371 resulting in a secondary defence where cells and virus become coated. Thus, any virus 372 particles having migrated to the cell interface are already inhibited to uptake. Likewise, the 373 formation of new viruses as a result of shedding, become incapacitated. This combinatorial 374 approach, coupled with the highly potent anti-viral capacity of the carrageenan towards 375 SARS-CoV-2, provides a powerful spray device with the capacity to prevent both contraction 376 and transmission. 377

Conclusions 378
As the primary mode of transmission for airborne viruses is uptake through the respiratory 379 tract, the nasal passage poses one of the largest risk factors to contraction. Although it is well 380 known that the nose filters 1000s of litres of air daily, there is little in the way of preventative 381 measures to ensure protection to infection. This study has demonstrated the formulation of a 382 potent antiviral nasal spray, with not only prophylactic capacity, but the ability to prevent 383 viral transmission. Its ability to completely inhibit infection is derived from the chemistry 384 (sulphated polymer backbone) of the active polymer, lcarrageenan. Spray characteristics 385 were engineered through the production of a composite, where a set of design rules were 386 understood to allow for manipulation over the material behaviours: spray coverage, viscosity 387 and yielding behaviour. Furthermore, understanding the role of each polymer in the 388 composite allowed for a preventative mechanism, using the synergy of both material and 389 antiviral properties to coat the biological interfaces, prevent viral uptake by host cells, and 390 eliminate through native clearance pathways. As such, this work presents a potential device 391 with the capacity to specifically target infection within the nasal cavity. 392 Gellan gum (CG-LA) was purchased from CP Kelco; TrypLE Express 1x was purchased 398 from Fisher Scientific; Black dye (Parker); Type 1 water (Milli-Q, Merck Millipore). 399

Materials and methods 393
Single-Component systems -colloidal suspensions were prepared through the addition of 400 polymer (0.2 to 1.0% (w/v)) to a dilute PBS (5% v) solution. Once added, the systems were 401 vigorously mixed and left to fully hydrate for 24 hrs. All samples were kept at ambient 402 temperature (ca. 20 °C) until further used.  All samples were kept at ambient temperature (ca. 20 °C) until further used. 408 Screening 409 Polymer screening was conducted using an airbrush (750 µm aperture) coupled to an oil-free 410 compressor (Badger, US), set to 1 bar. Test material (0.9 ml) was mixed with black dye (0.1 411 ml) and sprayed across an acetate sheet set to a 45° incline. The airbrush was then cleaned 412 using a succession of 70% ethanol and water. Spray distributions were visually analysed for 413 homogeneity and retention. 414

Rheology 415
Viscometric analysis was undertaken on a rotational rheometer (Kinexus Ultra, Netzsch 416 Geratebeu GmbH, DE) fitted with a cone and plate (4°, 40 mm diameter) geometry. Tests 417 were conducted at 25 °C, under stress control. Dynamic viscosity was analysed by reduction 418 of the shear stress from a maximum of 100 to 0.001 Pa (dependent on test material to prevent 419 expulsion from the gap at lower viscosities) over a 2 mins ramp time. Kinexus software was 420 used to characterise the flow profiles using both power law and Cross models. 421 ranks using the Mann-Whitney. Significance has been shown on plots using the following 469 notation: n.s -not statistically different; * -p<0.05; ** -p<0.01; and, *** -p<0.001. 470