WO2023235991A1 - Formulations and methods for protein encapsulation by spray freeze drying and microencapsulated compositions thereof - Google Patents

Abstract

Provided herein are formulations for spray freeze drying to encapsulate active ingredients, methods of preparing a composition of spray freeze dried micro-particles from the formulations described herein, and micro-particle compositions. In particular, the micro-particle compositions may be for use in the treatment or prophylaxis of a respiratory virus. The respiratory virus may be a coronavirus. In particular, the coronavirus infection may be selected from one or more of the following: Severe Acute Respiratory Syndrome (SARS) coronavirus-1 (SARS-CoV-1) infection; SARS coronavirus-2 (SARS-CoV-2) infection; and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) infection. More specifically, the coronavirus infection may be a human coronavirus 229E (HCoV-229E) infection. The active ingredients may include a protein or a fragment thereof, may be an angiotensin converting enzyme 2 (ACE2) or a fragment thereof. The protein encapsulating micro-particle composition may be used for the delivery of therapeutic proteins to the nasal mucosa.

Description

FORMULATIONS AND METHODS FOR PROTEIN ENCAPSULATION BY SPRAY FREEZE DRYING AND MICROENCAPSULATED COMPOSITIONS THEREOF

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/350,560 filed 9 June 2022 entitled "FREEZE SPRAY DRYING FOR PROTEIN ENCAPSULATION".

Technical Field

The present invention relates to methods for encapsulating and delivering active ingredients. In particular, herein are provided formulations for spray freeze drying to encapsulate active ingredients, methods of preparing a composition of spray freeze dried micro-particles from the formulations described herein, and micro-particle compositions encompassing the active ingredients. The micro-particle compositions for use in the treatment or prophylaxis of a respiratory virus are provided. The respiratory virus may be a coronavirus. In particular, the coronavirus infection may be selected from one or more of the following: Severe Acute Respiratory Syndrome (SARS) coronavirus-1 (SARS-CoV-1) infection; SARS coronavirus-2 (SARS-CoV-2) infection; and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) infection. More specifically, the coronavirus infection may be a human coronavirus 229E (HCoV-229E) infection. The active ingredients may include a protein or a fragment thereof, for example the protein or fragment thereof may be a receptor protein for a respiratory virus. The protein or fragment thereof may be an angiotensin converting enzyme 2 (ACE2) or a fragment thereof. The respiratory virus may be selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. The protein encapsulating micro-particle composition may be used for the delivery of therapeutic proteins to the nasal mucosa or other active site within a patient requiring treatment with said therapeutic protein. Background

Encapsulation and delivery of protein therapeutics is evolving towards improved efficiency of encapsulation and efficacy of the delivered proteins, which in turn has opened up a broad range of diseases for treatment [2,3]. However, more effort is needed to produce stable compositions comprising effective proteins for delivery. In particular, challenges associated with protein delivery are mostly related to the instability of the proteins, due to weak non- covalent atomic/group interactions, such as hydrophobic, electrostatic, hydrogen bonding, and van der Waals forces. The low stability of proteins is exacerbated by the high molecular weight and complex structure of some proteins [2], which can make encapsulation and delivery difficult. For most of the proteins, the inability to maintain their complex structures leads to denaturation, aggregation, or modification, which often results in a loss of activity [1]. Such delicate stability can be compromised by many factors, including metal ions, temperature, pH, and adsorption. Furthermore, protein instability often results in a relatively short half-life making protein therapies generally less effective.

Despite considerable effort, the most common way to deliver proteins is intramuscularly, but these methods are still considered invasive and challenging, especially for self- administration. Alternative protein delivery methods, such nasal, lung, or gastrointestinal, are usually achieved by protein encapsulation. During the process of delivery, the proteins are protected by an ‘enclosure’ shell and once the target is reached, the proteins are slowly released. The idea of encapsulating proteins in order to preserve their integrity and properties is derived from nature (for example, viral capsids). There are two main techniques used for micro-encapsulation, chemical and mechanical. The first technique has the advantage of high yield and high efficiency. For instance, solvent evaporation/extraction is a common method to produce, in large scale, peptide drugs with low aqueous solubility. However, chemical processes often have a low efficiency and are sensitive to temperature, type of polymer used, presence of impurities, etc. Mechanical processes are often easier to scale up, but are prone to producing polymorphic products. The most common mechanical process used for microencapsulation is spray drying. Spray drying is appealing for its broad applicability, but is prone to producing polymorphic products, is dependent on many variables, and damage can result from the high temperatures required for the atomizing procedure. The first two limitations could be overcome by careful selection of: solution viscosity; compounds weight percentage; drying air temperature; drying air flow rate; solution flow rate; etc. The high temperatures typically used in the atomizing process may be avoided by employing alternative techniques, like spray freeze drying [4]. With temperatures below 0°C, spray freeze drying can contribute to protein stability and usually includes the use of a cryogenic fluid to maintain the temperatures below a desired level. A cryogenic fluid may be used in the drying air or in the collection apparatus. Having the cryogenic fluid either fully or partially in the drying air, provides for a high level of control and can improve encapsulation efficiency [5]. However, to accommodate the cryogenic fluid particle formation process and the extreme low temperature at the atomizer, dryer designs can get quite complicated. A current variation of spray freeze drying is spray drying into a cryogenic fluid [6], where droplets are sprayed into liquid nitrogen (LN2). The nitrogen is left to dry and subsequent freeze drying is used to remove any residual water. The advantage of this technique is the ease of use and the production of micro-particles with a highly porous surface structure, for superior aerosol performance and having a favorable aerodynamic diameter [4].

A number of stabilizers may be routinely employed to preserve protein stability against stress-induced degradation, and may be divided into five broad groups: proteins (ex. BSA), amino acids (ex. glycine, alanine), polyols (ex. polyethylene glycol), carbohydrates (ex. glucose, lactose, sucrose, trehalose), and others (ex. surfactants, polymers, salts) [14]. Furthermore, the effect of formulation variables on particle size and stability have been studied [8]. Small molecule (i.e. indomethacin) microcapsules have been formed with polyvinylpyrrolidone (PVP) and stearic acid via a co-freeze-drying and ultrasound assisted spray-congealing process [7]. Similarly, lysozyme protein spray freeze dried spheres were produced with stabilizers such as bovine serum albumin, PVP or dextran with mannitol [5]. Alternative insulin protein spray freeze dried lyospheres were made from solutions containing PVP, mannitol and permeation enhancing excipients such as sodium taurocholate or cyclodextrin [13]. There are some attempts of generating guidelines on the use of spray drying in a cryogenic fluid [4,8]. A previous study analyzed the influence of the parameters of the spray dryer, such as drying air and liquid flow rates, to the morphology of spray freeze dried micro- particles. Here, we investigate the influence of the formulation compounds to the morphology of the micro-particles and to the stability of the encapsulated proteins.

Severe Acute Respiratory Syndrome coronavirus 2 [SARS-Cov-2] became the cause of the coronavirus disease 2019 [COVID-19] pandemic, which is reported to be responsible for 5 million deaths globally [15].

Numerous studies suggest that SARS-CoV-2 is an airborne virus, and that the most common form of transmission is through respiratory droplets or particles [16, 17, 18, 19]. Accordingly, the human nose is likely one of the main entry points for airborne pathogens [20]. In the human nose, angiotensin-converting enzyme 2 [ACE2], is reported to be a mediator for virus-cell fusion, and ACE2 is found in several epithelial, glandular, or basal cells, including the olfactory epithelium, the nasal septum, and the nasal conchae [21].

Due to the role of ACE2 in facilitating SARS-CoV-2 cell entry, decreasing or repressing the number of cell receptors for ACE2 is a potential SARS-CoV-2 treatment or prophylaxis [22]. Zeinalan et al. [23] attempted to use angiotensin receptor 1 [AT1R] blockers as therapeutics for reducing the aggressiveness and mortality from SARS-CoV-2 virus infections. However, these blockers seem to have counteracting effects and have been proven to increase the production of ACE2 in the heart [24]. Furthermore, the binding of viruses and the cell receptor resulted in a downregulation of ACE2, leading to heavy damage to the lungs. Higher ACE2 expression in SARS-CoV-2 infected patients that are treated with AT1R blockers, while paradoxical, may protect them against acute lung injury [25]. However, the use of AT1R blockers is problematic given that these treatments regulate high blood pressure, are used to treat heart failure, and prevent kidney failure, plus they have a common side effect of causing coughing [26].

Another option is to disrupt or mask the interaction between the spike proteins of SARS- CoV-2 and ACE2 receptors. Soluble ACE2 or sACE2 is produced by the heart and the lungs, while membrane bound ACE2 or mACE2 is found in the olfactory epithelium, the nasal septum, and the nasal conchae. Recombinant cell receptor Angiotensin-Converting Enzyme2 [rhACE2] is commercially available and can serve as a decoy to lure the virus to bind to the decoy instead of the mACE2 on the cell, and inactivating the virus before it can enter cells [27]. Some studies have explored the use of rhACE2 as a therapy for Covidl9 [28, 29, 30]. The most common route of delivery for rhACE2 is intravenous [IV], which is both highly invasive [31] and prone to potential side effects [32].

Furthermore, aqueous solutions of rhACE2 are likely to be prone to chemical instability resulting in diminished shelf-life. The use of dry powders could be a solution [33]. Moreover, the use of engineered dry powders can allow the use of a minimal quantity of otherwise expensive bioactive compounds, such as rhACE2, and still maintain effectiveness. Specifically, spray freeze drying [SFD] is the only spraying technique that allows control over the properties of sprayed powders at low temperatures [34].

Although the affinity of rhACE2 for the spike protein of SARS-CoV-2 has already been explored [31], and the idea of using rhACE2 as a possible treatment of Covidl9 has been published in a few research studies [30, 31, 35], an effective inhalable form of rhACE2 for delivery to the nasal cavity as prophylactic is yet to be realized.

Summary

The present invention is based, in part, on the surprising discovery that a particular polymer, polyvinyl pyrrolidone [PVP], and average molecular weight of at least 1,300 kDa is particularly useful in preparing a formulation for spray freeze drying to produce micro- particle compositions that encapsulate an active ingredient. The formulation for spray freeze drying comprising: [a] a polyvinyl pyrrolidone [PVP] polymer having an average molecular weight of at least 1,300 kDa; [b] one or more sugars or sugar alcohols; [c] one or more amino acids; and [d] an active ingredient. In particular, the active ingredient may be a protein or a fragment thereof. Furthermore, the protein or a fragment thereof may be at a concentration up to 10μg/ml. The resulting micro-particle compositions may encapsulate rhACE2 to produce a dry power between 100-200 μm in diameter and having good protein stability, and which may be suitable for nasal administration. Such micro-particle compositions encapsulating rhACE2 may be used for the treatment of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-COV2).

In a first embodiment, there is provided a formulation for spray freeze drying, the formulation for spray freeze drying including: (a) a polyvinyl pyrrolidone (PVP) polymer which may have an average molecular weight of at least 1,300 kDa; (b) one or more sugars or sugar alcohols; (c) one or more amino acids; and (d) an active ingredient.

The formulation may include the (a) a polyvinyl pyrrolidone (PVP) polymer which may have an average molecular weight of at least 1,300 kDa; (b) one or more sugars or sugar alcohols; (c) one or more amino acids; and (d) an active ingredient in solution that may be suitable for spray freeze drying.

The PVP polymer may have an average molecular weight of at least 1,000 kDa. The PVP polymer may have an average molecular weight of at least 900 kDa. The PVP polymer may have an average molecular weight of at least 800 kDa. The PVP polymer may have an average molecular weight of at least 700 kDa. The PVP polymer may have an average molecular weight of at least 600 kDa. The PVP polymer may have an average molecular weight of at least 500 kDa. The active ingredient may be a protein or a fragment thereof. The active ingredient may be a protein or a fragment thereof at a concentration up to 10μg/ml. The active ingredient may be an angiotensin converting enzyme 2 (ACE2) or a fragment thereof. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; sucrose; mannitol; xylitol; sorbitol; and trehalose. The one or more sugars or sugar alcohols may be at a concentration between 9-11 mg/ml. The one or more sugars or sugar alcohols may be mannitol and has a concentration between 9-11 mg/ml. The one or more sugars or sugar alcohols may be mannitol and lactose. The ratio of mannitoldactose may be 8:1.

The one or more amino acids may be a hydrophobic amino acid. The one or more amino acids may be selected from one or more of: leucine; tyrosine; alanine; isoleucine; lysine; histidine; threonine; cysteine; methionine; phenylalanine; tryptophan; and valine. The one or more amino acids may be selected from one or more of: leucine; alanine; isoleucine; lysine; histidine; threonine; cysteine; methionine; phenylalanine; tryptophan; and valine. The one or more amino acids may be selected from one or more of: leucine; glutamine; and asparagine. The one or more amino acids may be selected from one or more of: leucine; and glutamine. The one or more amino acids may be selected from one or more of: leucine; tri- leucine; arginine; glutamine; and tyrosine. The one or more amino acids may have a concentration between 2-2.2 mg/ml. The one or more amino acids may be leucine and has a concentration between 2-2.2 mg/ml. Alternatively, the one or more amino acids may be within the weight percentage range of 15% - 25%.

The PVP may have a concentration between 6-7 mg/ml. The PVP may have a concentration of 6 mg/ml.

In a further embodiment, there is provided a micro-particle composition, the micro-particle composition including: (a) a PVP polymer which may have an average molecular weight of at least 1,300 kDa; [b] one or more sugars or sugar alcohols; (cj one or more amino acids; and (d) an active ingredient.

The micro-particle composition, the micro-particle composition including: (a) a PVP polymer which may have an average molecular weight of at least 1,300 kDa; [b] one or more sugars or sugar alcohols; (cj one or more amino acids; and (d) an active ingredient, may be the resulting product of the spray freeze dried formulation described herein.

The PVP polymer may have an average molecular weight of at least 1,000 kDa. The PVP polymer may have an average molecular weight of at least 900 kDa. The PVP polymer may have an average molecular weight of at least 800 kDa. The PVP polymer may have an average molecular weight of at least 700 kDa. The PVP polymer may have an average molecular weight of at least 600 kDa. The PVP polymer may have an average molecular weight of at least 500 kDa. The micro-particle may have a diameter between 100-200 μm. The micro- particle may have a projected area equivalent diameter [d_a] of about 100 μm to about 200 μm. The active ingredient may be a protein or a fragment thereof. The active ingredient may be a decoy protein or a fragment thereof which binds to a respiratory virus for use in the treatment or the prophylaxis of a respiratory virus infection. The active ingredient may be an ACE2 protein or a fragment thereof. The respiratory virus may be a coronavirus. In particular, the coronavirus infection may be selected from one or more of the following: Severe Acute Respiratory Syndrome (SARS) coronavirus-1 (SARS-CoV-1) infection; SARS coronavirus-2 (SARS-CoV-2) infection; and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) infection. More specifically, the coronavirus infection may be a human coronavirus 229E (HCoV-229E) infection. The active ingredient may be a protein or a fragment thereof and may be present at up to 10μg per micro-particle. The micro-particle may be suitable for nasal administration. The micro-particle may be suitable for pulmonary administration. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; sucrose; mannitol; xylitol; sorbitol; and trehalose. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; sucrose; mannitol; xylitol; and sorbitol. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; sucrose; mannitol; and xylitol. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; sucrose; and mannitol. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; glucose; and mannitol. The one or more sugars or sugar alcohols may be selected from one or more of: lactose; and mannitol. The one or more sugars or sugar alcohols may be mannitol.

The one or more sugars or sugar alcohols may be mannitol and may be between about 49.45 % and about 57.89 % by weight of the final composition. The one or more sugars or sugar alcohols may be between about 49.45 % and about 57.89 % by weight of the final composition. The one or more sugars or sugar alcohols may be between about 49 % and about 58 % by weight of the final composition. The ratio of mannitol:lactose may be 8:1.

The one or more amino acids may be leucine and may be between about 10.00 % and about 12.79 % by weight of the final composition. The one or more amino acids may be between about 10.00 % and about 12.79 % by weight of the final composition. The one or more amino acids is within the weight percentage range of 15% - 25%. The one or more amino acids may be leucine and may be between about 10.00 % and about 12.79 % by weight of the final micro-particle composition. The one or more amino acids may be between about 10.00 % and about 12.79 % by weight of the final micro-particle composition. The one or more amino acids is within the weight percentage range of 15% - 25%.

The PVP may be between about 31.25 % and about 38.89 % by weight of the final composition. The PVP may be between about 31% and about 39% by weight of the final composition. The PVP may be between about 31.25 % and about 38.89 % by weight of the final micro-particle composition. The PVP may be between about 31% and about 39% by weight of the final micro-particle composition.

In a further embodiment, there is provided a method of preparing a composition of spray freeze dried micro-particles, the method including: (a) spraying a liquid formulation including: [i] a PVP polymer having an average molecular weight of at least 1,300 kDa; (ii) one or more sugars or sugar alcohols; (iii) one or more amino acids; and [iv] an active ingredient; to form droplets by atomization; [b] freezing the droplets in a cryogenic fluid to form powdered micro-particles; and (cj removing residual moisture from the powdered micro-particles by freeze drying in a vacuum chamber.

The liquid formulation may be as described herein. The spraying may have a flow rate of about 5 ml/minute or less. The freeze drying may be for 4 hours at 300 mTorr and 20 hours at 100 Torr. The freeze dried powdered micro-particles may be an average diameter of about 100 μm to about 200 μm. The freeze dried powdered micro-particles may be suitable for intranasal administration.

In a further embodiment, there is provided a use of the micro-particle composition described herein, for the treatment of a viral respiratory infection.

In a further embodiment, there is provided a method of treating a viral respiratory infection, comprising administering a micro-particle composition described herein, to a subject in need thereof. The micro-particle composition described herein for the treatment a viral respiratory infection.

In a further embodiment, there is provided a use of the micro-particle composition described herein, for intranasal delivery of an active ingredient. In a further embodiment, there is provided a method for intranasal delivery of an active ingredient, comprising administering a micro-particle composition described herein to a subject in need thereof. The micro-particle composition described herein for intranasal delivery of an active ingredient.

Herein is described protein encapsulating formulations and methods for preparation of said formulations. Such formulations may be used to deliver a protein to the desired site of action including the delivery of therapeutic proteins within a patient.

In another embodiment, there are provided protein encapsulating formulations comprising the active ingredient and a number of excipients as described below; An active ingredient, A polymer, One or more sugars or sugar alcohols and One or more amino acids.

In another embodiment, there are provided protein encapsulating formulations that produce atomized micro-particles of preferably 100-200 μm in diameter.

In another embodiment, there are provided protein encapsulating formulations wherein the active ingredient is a protein and preferably proteins at a concentration of up to 10μg/ml

In another embodiment, there are provided protein encapsulating formulations wherein the protein is a therapeutic protein, preferably a receptor protein for respiratory viruses such as Severe Acute Respiratory Syndrome coronavirus 2 (SARS-COV2).

In another embodiment, there are provided protein encapsulating formulations wherein the therapeutic protein is angiotensin converting enzyme 2 (ACE2] or fragments thereof.

In another embodiment, there are provided protein encapsulating formulations wherein the polymer is a polyvinylpyrrolidone [PVP], preferably PVPs having a molecular weight of at least 1300 kDa.

In another embodiment, there are provided protein encapsulating formulations wherein the sugar or sugar alcohol may include one or more of either mannitol or lactose. Wherein if the formulation comprises both mannitol and lactose the ratio of mannitol: lactose is preferably at least 8:1. In another embodiment, there are provided protein encapsulating formulations wherein the amino acid may include one or more of either leucine, tri-leucine, tyrosine or arginine. The formulations comprise one or more amino acids preferably within the weight percentage range of 15-25 wt.%.

In another embodiment, there are provided protein encapsulating formulations comprising PVP, mannitol and leucine at weight percentages of 60%, 90% and 20%, respectively. In such formulations the total weight of PVP is preferably in the range of 6-7 mg/ml; mannitol preferably in the range of 9-llmg/ml and leucine preferably in the range of 2-2.2 mg/ml. The active ingredient in such formulations may include proteins and preferably therapeutic proteins such as ACE2.

In another embodiment, there are provided methods for preparing protein encapsulating formulations by spray freeze drying comprising the following steps; Preparation of a solution comprising a protein encapsulating formulation as described herein, Spray atomization of the solution into a cryogenic liquid, preferably liquid nitrogen, to form powdered micro-particles of protein encapsulated in a polymer shell then, Removal of residual moisture from said powder by freezing at -80°C followed by freeze-drying in a vacuum chamber.

In another embodiment, there are provided methods for preparing protein encapsulating formulations that produce atomized micro-particles of preferably 100-200 μm in diameter.

In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the active ingredient is a protein and preferably proteins at concentrations of up to 10 μg/ml.

In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the protein is a therapeutic protein, preferably a receptor protein for respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-COV2).

In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the therapeutic protein is angiotensin converting enzyme 2 (ACE2) or fragments thereof. In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the polymer is a polyvinylpyrrolidone (PVP), preferably PVPs having a molecular weight of at least 1300kDa.

In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the sugar or sugar alcohol may include one or more of either mannitol or lactose. Where the formulation comprises both mannitol and lactose the ratio of mannitol: lactose is at least 8:1.

In another embodiment, there are provided methods for preparing protein encapsulating formulations wherein the amino acid may include one or more of either leucine, tri-leucine, tyrosine or arginine. The formulations comprise one or more amino acids preferably within the weight percentage range of 15-25 wt.%.

In another embodiment, there are provided methods for preparing protein encapsulating formulations comprising PVP, mannitol and leucine at weight percentages of 60%, 90% and 20%, respectively. In such formulations the total weight of PVP is preferably in the range of 6-7 mg/ml; mannitol preferably in the range of 9-llmg/ml and leucine preferably in the range of 2-2.2mg/ml. The active ingredient in such formulations may include proteins and preferably therapeutic proteins such as ACE2.

In another embodiment, there are provided methods of treatment using protein encapsulating formulations as described herein delivered to the nasal mucosa or other active site within a patient requiring treatment with a therapeutic protein.

In another embodiment, there are provided methods of treatment using protein encapsulating formulations as described herein delivered to the nasal mucosa or other site within a patient requiring treatment with a receptor protein for respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS COV-2).

In another embodiment, there are provided methods of treatment using protein encapsulating formulations as described herein delivered to the nasal mucosa or other site within a patient requiring treatment with recombinant human angiotensin converting enzyme 2 (rhACE-2) or fragments thereof. Brief Description of Drawings

Figure 1A shows a schematic of the procedure for making spray freeze dried particles to encapsulate an active ingredient.

Figure 1B shows the preferred parameters for spray freeze drying (SFD).

Figure 2 shows the projected ratio between the project area equivalent diameter (d_a) and the total weight percentage (wt %) a) and the ratio between the carrier, mannitol or lactose, and the polymer (r) b). The scale bars for the images shown per each case are 500 μm and 50 μm for the low and high magnification pictures, respectively. For all cases shown above, the weight percentage of the amino acid, leucine, is 20%.

Figure 3 shows the trend in projected area equivalent diameter for an increase in ratio between amino acid and carrier (mannitol) r_a, and for an increase in ratio between two carriers, mannitol and lactose, r_c. For both plots a) and b), the total weight percentage is 171 and the ratio between the sum of the carriers and amino acid and the polymer is 1.8.

Figure 4 shows the impact of the molecular weight of different proteins, a), and of different polymers, b), to the size of spray freeze dried micro-particles

Figure 5 shows the impact of the ratio between trileucine and leucine, top image, and of types of amino acid, bottom, to the size of spray freeze dried micro-particles

Figure 6 shows fluorescence images of different layers along one axis of spray freeze dried micro-particles. The cases a, b, c, and, d relate to the cases involving a total weight percentage of 231 and a ratio between the carrier, mannitol, and the polymer, PVP 1300, of 0.61, a); a total weight percentage of 171 and a ratio between the carrier, mannitol, and the polymer, PVP 1300, of 0.66 b); total weight percentage of 171, a ratio between the carrier, mannitol, and the polymer, PVP 1300, of 0.63. In this case, the ratio between the amino acid, leucine, and the carrier, mannitol is decrease to 0.16 with respect to the other cases showing a ratio of 0.2 c) a total weight percentage of 171 and the ratio between the carrier, mannitol and lactose, and the polymer, PVP 1300, of 0.66. In this case, the ratio between mannitol and lactose is 8 d). Figure 7 shows a), the deconvolution of the amide I of bovine serum albumin (BSA) is shown; in b to h), the percentage difference between the areas of β-sheet, α-helix, β-turn, and β-antiparallel of the BSA spectrum and the spectra of samples differing in total weight, ratio of amino acid and carrier, ratio of carrier and polymer, ratio of two carriers, polymer molecular weight, amino acid, and protein, respectively, is shown.

Figure 8 shows a comparison between the same case (5) spray freeze dried by using a feeding flowrate of 15 ml/min a) and of 5 ml/min b).

Figure 9 shows a comparison of the effect of different freeze-drying procedures. On the left- hand side, the freeze-drying procedure was 24 hours at 750 mBar a); on the right-hand side, the freeze-drying procedure was 4 hours at 300 mTorr followed by 20 hours at 100 mTorr b).

Figure 10 shows a), morphology and projected equivalent area diameter of spray freeze dried micro-particles containing rhACE2. In b), distribution of rhACE2 within sprays freeze dried micro-particles, in c) and d), morphology and projected equivalent diameter of spray freeze dried micro-particles obtained with different solutions including trileucine, trehalose, and alternative components.

Figure 11 shows in the top row, image of fluorescent rhACE2 (left) and of the distribution of fluorescent rhACE2 within spray dried micro-particles (right), at the bottom row, the XPS analysis of the distribution of the chemical compounds in spray freeze dried micro-particles.

Figure 12 shows FTIR analysis of spray freeze dried micro-particles. In a), the deconvolution of the peak between 1600 and 1750 cm^-1, in b), the whole IR spectrum of rhACE2, in c), d), e), and f) the difference of the deconvoluted peak with respect to the peaks of free rhACE2 are shown for an increase of rhACE2, in total weight percentage, in the ratio between trileucine and leucine, and ratio between trehalose and leucine, respectively.

Figure 13 shows HPLC analysis of freeze dried micro-particles containing rhACE2. The difference between the areas of the main peaks obtained for freeze dried micro-particles and free rhACE2 are shown. Figure 14 shows ¹H NMR analysis of pure rhACE2, left-hand side, and of spray freeze dried micro-particles from a formulation with 9.0 mg/ml, 6.0 mg/ml, and 2.2 mg/ml, for PVP, mannitol, and leucine, respectively, and 40 μg/ml of rhACE2, at the right-hand sides.

Figure 15 shows ELISA analysis of spray freeze dried micro-particles from a formulation with 9.0 mg/ml, 6.0 mg/ml, and 2.2 mg/ml, for PVP, mannitol, and leucine, respectively, and different quantities of rhACE2

Figure 16 shows binding efficiency, indicated as the difference in percentage between the calibration curve at a certain quantity and the ACE2 samples, of the cases containing concentrations (mg/ml) of 9.0, 6.0, and 2.2 for PVP, mannitol, and leucine, and quantities of ACE2 of 2, 5, 10, 20, 30, and 50 μg/ml.

Detailed Description

The following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown. Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention.

As described herein there are provided protein encapsulating formulations, methods for preparation of micro-particle compositions using said formulations, and micro-particle compositions for the delivery of active ingredients to the desired site of action.

As used herein, polyvinylpyrrolidone (PVP) is a polymer used in formulations described herein and for preparing micro-particle compositions as described herein. PVP having a molecular weight M_w of at least 1300 kDa is suitable and preferred, but PVP 360 (360 kDa) can also be used and PVP 55 (55 kDa) was also tested but showed poor morphology and encapsulation of protein. PVP 1300 (M_w ~1, 300, 000 Da (CAS No.: 9003-39-8), is commercially available under product number 437190 from Sigma Aldrich™. A PVP having a molecular weight M_w of at least 1000 kDa would be suitable for formulations and methods described herein for making micro-particle compositions. Polyvinylpyrrolidone abbreviated as PVP, is also known as polyvidone or povidone. PVP is a water-soluble polymer made from the monomer N-vinylpyrrolidone. PVP is available in a broad range of molecular weights. As described herein, PVP has a molecular weight M_w preferably greater than 1300 kDa.

As used herein, carriers refer to sugars or sugar alcohols. Two of the carriers employed were: D-Mannitol ((CAS No.: 69-65-8) M4125, Sigma Aldrich™); Lactose (61345, Sigma Aldrich™); and Trehalose (T9449 D-(+)-Trehalose dihydrate, Sigma Aldrich™). Also, the carriers may be used alone or in combination as described herein. As used herein sugar means soluble carbohydrates such as monosaccharides, disaccharides and polysaccharides and commonly exemplified by glucose and sucrose. As described herein the sugar is preferably lactose. As used herein sugar alcohol (also called polyhydric alcohols, polyalcohols, alditols or glycitols) refers to organic compounds derived from sugars, containing one hydroxyl group (-OH) attached to each carbon atom. Sugar alcohols are often used as artificial sweeteners and is exemplified by xylitol and sorbitol. As described herein, the sugar alcohol is preferably mannitol.

As used herein, excipients refer to amino acids. Several amino acids were also tested as excipients: Leucine (L-leucine, ((CAS No.: 61-90-5) L80000, Sigma Aldrich™); trileucine (L0879, Sigma Aldrich™); L-Arginine (A5006, Sigma Aldrich™); L-Glutamine (G7513, Sigma Aldrich™); and L-Tyrosine (T3754, Sigma Aldrich™). Also, the excipients may be used alone or in combination as described herein. As used herein amino acid refers to organic compounds that contain amino and carboxyl functional groups, along with a side chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, although other elements are found in the side chains of certain amino acids. Some amino acids are used in the biosynthesis of proteins. As described herein, amino acids are preferably leucine, tri-leucine, tyrosine, glutamine or arginine.

As used herein, spray freeze drying means the process of drying a material involving a solution being atomized, solidified and sublimed at low temperature. The atomized material is typically solidified by rapid freezing into a cryogenic fluid such as liquid nitrogen (LN2) along with additional excipients that protect the structure, activity and stability of said material. The mixture then undergoes further drying by freeze-drying in a vacuum chamber to remove residual moisture. As described herein, formulations for preparation of proteins by spray freeze drying, which are normally sensitive to degradation by heat, moisture or chemical/ enzymatic action are provided. This is of particular concern when preparing therapeutic proteins that requires its structure and/or biological activity to be preserved until it is delivered to the desired site of action.

As used herein "receptor protein” for "respiratory virus(es)” means a protein normally present on the surface of host cells that is bound by a viral spike protein and required for the entry of the virus into the host cell. The viral receptors for many respiratory viruses have been identified. As described herein the receptor protein may be the viral receptor for severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), which has been identified as angiotensin converting enzyme 2 (ACE-2). As used herein, the term "Coronavirus” abbreviated CoV refers to a family of enveloped, positive-sense, single-stranded, and highly diverse RNA viruses, with four distinct groups (i.e. alpha, beta, gamma, and delta). The α- coronavirus and β-coronavirus are of particular interest, because of their ability to cross from non-human animals to humans. So far, there are seven documented human coronaviruses (hCoVs), including the beta-genera CoVs, namely Severe Acute Respiratory Syndrome (SARS)-CoV (SARS-CoV), Middle East Respiratory Syndrome (MERS)-CoV (MERS- CoV), SARS-CoV hCoV-HKU1, and hCoV-OC43 and the α-genera CoVs, which are hCoV-NL63 and hCoV-229E, respectively.

As used herein angiotensin converting enzyme 2 (ACE-2) means a zinc- containing metalloenzyme located on the surface of endothelial and other cells. ACE-2 is an enzyme and a negative regulator of the renin-angiotensin system (RAS) and lowers blood pressure by catalyzing the hydrolysis of angiotensin II (a vasoconstrictor peptide) into angiotensin (1-7) (a vasodilator). The human version of this enzyme is referred to as hACE-2. As described herein ACE-2 may include hACE-2, recombinant human ACE-2 and also any fragments of hACE-2 or rhACE-2 which are able to bind the viral spike protein of SARS-COV-2. As described herein ACE2 and fragments thereof may be considered active ingredients.

An "effective amount” of an active ingredient as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time as needed, to achieve the desired therapeutic result, such as reduced tumor size, increased life span or increased life expectancy. A therapeutically effective amount of an active ingredient may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the active ingredient to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active ingredient are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as smaller tumors, increased life span, increased life expectancy or prevention of the respiratory viral infection. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active ingredient(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As specifically described herein most of the micro-particle compositions are tailored for nasal inhalation. In general, active ingredients, as described herein, should be used without causing substantial toxicity. Toxicity of the active ingredients as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD₅₀ (the dose lethal to 50% of the population) and the LD₁₀₀ (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be appropriate to administer substantial excesses of the active ingredients. Some active ingredients as described herein may be toxic at some concentrations. Titration studies may be used to determine toxic and non-toxic concentrations. Animal studies may be used to provide an indication if the active ingredients have any effects on other tissues.

An active ingredient, as described herein, may be administered to a subject. As used herein, a "subject” may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a viral infection, such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV. Alternatively, the infection may be a coronavirus infection. Diagnostic methods for various viral infections, are known to those of ordinary skill in the art.

Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art.

Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention. Methods and materials

Formulations

As a readily available protein, for testing the encapsulation efficiency of spray drying in a cryogenic fluid we used Bovine Serum Albumin (BSA, 9048-46-8, VWR Chemicals™). For fluorescence analysis, fluorescent BSA was used (Albumin from Bovine Serum (BSA), FITC conjugate, A23015, ThermoFisher™). Other proteins were used to verify the encapsulation efficiency of this procedure utilizing a variety of proteins: Pea protein and whey protein isolate (Canadian protein™), soy protein (Red Mill™), Hemoglobin from bovine blood (H3760, Sigma Aldrich™), β-Lactoglobulin from bovine milk (L3908, Sigma Aldrich™). Furthermore, a potential Covid-19 protein treatment for nasal inhalation was tested, in the form of recombinant human Angiotensin-Converting Enzyme2 (rhACE2 - 10108-H08B, Sino Biological™). A polymer was used for generating a micro-particle shell during the particle formation process: Polyvinylpyrrolidone (PVP) 1300 (M_w ~ 1,300,000 Da (CAS No.: 9003-39- 8), 437190, Sigma Aldrich™). As a comparison, the same polymers with different molecular weights were also used (PVP 360 and PVP 55, Sigma Aldrich™). Two of the carriers employed were: D-Mannitol ((CAS No.: 69-65-8) M4125, Sigma Aldrich™) and Lactose (61345, Sigma Aldrich™). Alternatively, Trehalose (T9449 D-(+)-Trehalose dihydrate, Sigma Aldrich™) could be used as a carrier. Several amino acids were also tested as excipients: Leucine (L-leucine, ((CAS No.: 61-90-5) L80000, Sigma Aldrich™); trileucine (L0879, Sigma Aldrich™); L-Arginine (A5006, Sigma Aldrich™); L-Glutamine (G7513, Sigma Aldrich™); and L-Tyrosine (T3754, Sigma Aldrich™).

The use of L-leucine and D-Mannitol are known to promote some particle formation [55, 56] and L-Leucine is known to enhance permeation through the nasal mucosa of different bioactive compounds [57, 58]. High molecular weight polymers are also known to assist with the formation of a shell early in the particle formation process [34, 43, 59].

When measuring the molecular weight of linear polymers, the individual polymer chains rarely have exactly the same degree of polymerization and molar mass. Accordingly, there is some distribution around an average value. The molar mass distribution of a polymer may be narrowed through polymer fractionation, but there is still going to be a range of molecular weights. The molecular weight of a polymer may be determined by a number of methods, number average molar mass [M_n]; mass average molar mass [M_w], is the weight average or weight average molecular weight; Z-average molar mass [M_z], where z stands for centrifugation (German Zentrifuge]; or viscosity average molar mass [M_v]. When Molecular weights are referred to herein, this is a mass average molar mass M_w, unless otherwise stated.

Briefly, the develoμment of the formulation involved three steps: dissolution of PVP polymer in water, the addition of mannitol and leucine, and aggregation of rhACE2. A solution of PVP and water was created using a microbalance (CP2245 Sartorious™]. D-Mannitol and L- Leucine were added to the solution, and a stirring at 100 rμm followed for 2 hours and shaking with an incubator shaker C25KC Incubator shaker (New Brunswick Scientific™) for 3 hours. Protein was added and the solution was stored at 4°C for 24 hours.

A preferred weight percentage for PVP 1300, D-Mannitol, and L-Leucine was 35, 52, and 13 %, respectively. These percentages were selected based on previous results [56, 60, 61]. The selected quantities of rhACE2 are: 0.5, 1, 2, 5, 10, 20, 40, and 70 μg/ml. The selected total weight of the initial formulation calculated in g/100 ml of 145, 155, 165, 175, 185, 195, 200, 210, and 220 was analyzed. For the last formulation, the quantity of rhACE2 was maintained stable at 2 μg/ml.

Spraying technique

The technique used to produce the dry powder was spray freeze drying [SFD]. SFD was selected since low temperatures were expected to generate small or null damage to rhACE2 [62]. Briefly, the formulation was sprayed through a 0.7 mm nozzle in a lab-scale spray dryer (Buchi 290™]. The drying parameters were 5 ml/min for feeding liquid flow, and 3 L/min for airflow, but 15 ml/min for feeding liquid flow was also tested. Higher feeding flowrates generally result in larger droplets and, thus, possibly larger micro-particles. Once frozen in the liquid nitrogen [LN2], these large micro-particles usually contain a higher amount of water within the formed shell. The water, when evaporated during the freeze-drying procedure, can burst a thin shell. Having a lower feeding flow rate generally results in micro- particles, having a lower water content and sometimes a thicker shell. These two properties can increase the resistance of the formed shell when exposed to the freeze-drying procedure. The sprayed droplets were collected into a Cylindrical Form Borosilicate Glass Dewar Flask (250 ml, StonyLab™) filled with liquid nitrogen (LN2). The cylinder was placed 10 cm below the atomizer (FIGURE 1A and B). Larger distances between the atomizer and the container of liquid nitrogen often result in a lower collection yield. When distances are greater than 10 cm, the collection area is smaller than the area of the spraying jet.

The slurry stored at -80 °C for 12 hours was later placed in a freeze dryer (Labconco Freezone 6™) to remove any remaining water. Various freeze drying times and pressures were tested. For example, (1) 48 hours at 300 mTorr and -3 ± 4 °C for 3 hours and at 100 mTorr and 8 ± 4 °C for 16 hours [62, 63]; (2) 300 mTorr at -10±2 °C for 4 hours and at 100 mTorr and 10±5°C for 16 hours; (3) 300 mTorr at -10±2 °C for 4 hours and at 100 mTorr and 10±5°C for 20 hours and (4) 24 hours at 750mBar. Different conditions have been shown to impact the morphology of the spray freeze dried micro-particles.

Morphological properties

The morphology of the SFD micro-particles was obtained by using a Scanning Electron Microscope (Hitachi S4700 SEM™, Ultrahigh-resolution SEM with field-emission gun). In order to prepare the samples for the morphological analysis, about 1 mg of material was placed on an Isopore™ membrane filter (13 mm of diameter an 0.4 μm pore size), which are positioned on an SEM stub. A layer of 8 nm [or 16nm] of gold was placed over each sample at 10 kV and 8 mA using a Cressington Sputter Coater™. The images obtained using the SEM were analyzed using Image]™ to derive the projected area equivalent diameter (da) [44], an indication of the diameter of the micro-particles. When determining the d_a, measurements of the diameter of 300 to 600 micro-particles were performed [34].

Due to the inherent fluorescence of rhACE2 [64], a confocal microscope (Olympus FV1000™ Laser Scanning/Two-Photon Confocal Microscope) was used for a qualitative analysis of the distribution of rhACE2 on the micro-particles. The wavelength used for the analysis was 527 nm. This analysis was conducted on powders obtained from four formulations varying in content, 1, 2, 10, and 20 μg/ml. For semi-quantitative analysis, X-ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos Analytical Axis ULTRA™ spectrometer containing a DLD spectrometer with a monochromatic aluminum source (AlKα, 1486.6 eV) operating at 150 W (10 mA emission current and 15 kV HT). Analysis was performed on a 700 x 300 μm² area of the sample. High-resolution scans were attained at a 100 meV step size, averaged over 3 scans, and pass energy of 20 eV. The ISO 15472 was used as a calibration procedure.

Chemical stability

The rhACE2 was analyzed by Attenuated total reflectance (ATR) - FTIR (Fourier-transform infrared spectroscopy - Spectrum 100, PerkinElmer™) and High-performance liquid chromatography (HPLC) was used. The spectra were deconvoluted using OriginPro™ deriving peak areas. The IR peaks of rhACE2 have not been identified in previous references. However, the typical peaks of other similar proteins are well-known. For instance, Amide I and amide II bands are located in the region 1600-1750 cm^{-1 51}. An alteration in intensity or position of these sub-peaks could indicate dimers or trimer of protein, which indicates aggregation of the protein and thus micro-particle degradation [66].

To verify the chemical stability data obtained from FTIR, the HPLC system (Agilent 1100™ series, Agilent™, Santa Clara, California, USA) was used. The HPLC contained a quaternary pump, an autosampler, a column oven, and a DAD detector. rhACE2 was analyzed by C18 column (Zorbax™, 3.5 μm, 4.6 mm x 150 mm, Agilent™, USA) at the wavelength of 210 nm. The mobile phase was acetonitrile: water containing 0.1% of trifluoroacetic acid (TFA) in a gradient ratio from 10/90 v/vto 100/0 for 10 min running. The mobile phase was pumped at a 1.0 ml/min flow rate. The column temperature was set to 25 °C.

Encapsulation efficiency

HPLC was used to measure the encapsulation efficiency. Samples were dispersed in water with the same weight percentage (3 wt %), placed in a filter (28-9323-19, GE Healthcare™, 100KDa), and centrifuged at 1000 rμm for 2 hours. The differences in peak area, measured with the HPLC, of filtered and non-filtered solutions indicate the encapsulation efficiency [67]. Binding affinity

The affinity between the rhACE2 and the spike proteins of SARS-CoV-2 (Sino Biological™, Spike S1+S2 ECD, A899P) was analyzed through the ELISA technique [68].

Cell line maintenance

The RPMI-2650 cell line (CCL-30) was purchased from the American Type Cell Culture Collection™ (ATCC). Cells were maintained in Minimum Essential Medium (MEM; Gibco™, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Invitrogen™) and 1% (v/v) non-essential amino acids (Sigma-Aldrich™) at 37 °C with 5% CO₂. All experiments utilizing RPMI-2650 cells were performed at least in triplicates.

Air-liquid interface (ALI) model

An ALI cell culture model was established using RPMI-2650 cells as previously described [52]. Briefly, cells were seeded (2.5 x 10⁶ cells/mL) onto the apical chamber of Snapwell™ inserts (Corning Costar™, USA) and incubated for 24 h. Following incubation, media on the apical chamber of the Snapwell™ inserts was aspirated. Media in the basolateral chamber was replaced with fresh media every two days for 14 days until confluence to establish tight junction formation by the ALI model and mimic the human nasal epithelial barrier.

Cytotoxicity ofrhACE2 drug

The cytotoxicity of rhACE2 formulation was determined on the RPMI-2650 cell line using an MTS Cell Proliferation Assay Kit (Promega™, USA) following manufacturer instructions [69]. Briefly, cells were seeded (2.4 x 10⁵ cells/cm²) on 96-well tissue culture plates (Corning Costar™). After 24 h of incubation, drug treatments were added with a concentration of drugs ranging from 0.0007813 to 1 mg/mL of rhACE2 drug formulation 9 resuspended in cell culture media and incubated for 24 h. MTS reagent was added to the cells and incubated for 4 h, and samples were read using a spectrophotometer (SpectraMax M2™, Molecular Devices™) at 490 nm. Cell viability for treated cells was determined in comparison to untreated cells. Transepithelial electrical resistance (TEER)

TEER was measured as described previously [70]. Briefly, TEER was measured using an EVOM2™ epithelial volt-ohm meter [World Precision Instruments™, USA]. TEER was measured once before drug exposure to the cells another time after drug deposition, followed by 4 h incubation at 37 °C, 5% CO₂, and 95% humidity.

Paracellular permeability of the ALI cell model

The epithelial tight junction [TJ] barrier function and apparent paracellular permeability of ALI-cultured RPMI-2650 cells exposed to the rhACE2 drug formulation were investigated using the sodium fluorescein permeability assay. Briefly, 200 μL of a 2.5 mg/mL sodium fluorescein [Sigma Aldrich™] solution in Hanks' Balanced Salt Solution [HESS] was added to the apical chamber, and 2 mL of pre-warmed HESS was added to the basolateral chamber. The cells were then incubated for 4 h at 37 °C with 5% CO₂, with 200 μL of basolateral samples collected every 15 min for 1 h to measure the rate of transport [flux] of the sodium fluorescein from the apical chamber to the basolateral chamber of the ALI culture. For analysis, the collected basolateral samples were diluted [1:100] and fluorescence was measured using the SpectraMax M2™ plate reader [excitation: 485 nm; emission: 538 nm]. The calculation of the permeation coefficient [Papp] was recorded according to the provided equation [1], whereby, dQ/dT represents the mass flux of sodium fluorescein [μg/s] across the epithelium, Co is the initial donor concentration [μg/mL], and A is the surface area of the Snap well membrane [cm²].

[1] Papp= [dQ]/[dT*C₀*A] rhACE2 drug formulation transport studies

The transport of rhACE2 Formulation 9 across the ALI model of RPMI-2650 cells was further assessed. Briefly, 3 mg of the dry powder formulation was loaded into the Unit Dose inhaler device [Aptar Pharma™, USA] and released into a modified nasal expansion chamber [52]. This 3D printed chamber has a 2 L volume, similar to the glass expansion chamber recommended by the FDA for testing nasal drug formulations [71]. Moreover, it allows the integration of Snapwell™ inserts cultured with RPMI-2650 cells inside the chamber to simulate the in vivo nasal drug deposition. The modified expansion chamber incorporated with ALI cultured RPMI-2650 cells was connected to the next generation impactor (Westech W7™, Westech Scientific Instruments™, UK) to mimic the human intranasal inhalation route. The Unit Dose inhaler was actuated at a 45° angle while a 15 L/min air flow rate was applied by using a vacuum pump (Westech Scientific Instruments™, UK) and a calibrated flowmeter. The actuation was performed within 4 s. Once the drug had been administered to the cells, 2 mL of HESS was added to the basolateral chamber of the Snapwell™ inserts and the cells were incubated at 37 °C, 5% CO₂, and 95% humidity for 4h. During this time, 200 μL samples were taken from the basolateral chamber every 30 min for the first 3 h and then every hour for the final 1 h, with samples being replaced by 200 μL of fresh pre-warmed HESS. After 4 h, the apical chamber was washed twice with HESS using a pipette to collect any residual drug on the cell layer. The cell layer was then scraped from the insert membrane and lysed using CelLyticTM buffer (Invitrogen™) to quantify the rhACE2 amount inside the cells. The amount of ACE2 in each sample collected at the different time points was determined using High-Performance Liquid Chromatography (HPLC).

Statistical approach

Each experiment was reproduced three times. Averages have been reported, and standard deviation and errors have been obtained by considering the differences between triplicates. Origin Pro™ was used for regression analysis and analysis of variance (ANOVA). p-Values of less than 0.05 were examined to be statistically significant.

Formulation matrix

To verify the impact of each component to the physical and chemical properties of the micro- particles achieved via spray freeze drying, a broad range of solutions was created, as shown in Error! Reference source not found.A. With the following matrix, the impact of the total weight percentage, the ratio between carrier and polymer (r), the ratio between amino acid and carrier (r_a), the ratio between two carriers (r_c), the molecular weight of the polymer and of the protein, and a change in amino acid is verified. Due to a short window (only 10 cm) for the shell to be formed before entering into the liquid nitrogen, highly concentrated (> 146 weight percentage) solutions were used.

Formulation matrix for ACE-2

In order to verify the impact of each component to the physical and chemical properties of encapsulation for rhACE2 for nasal delivery via spray freeze drying, a broad range of solutions were implemented, as shown in Table 1B.

Table 1B List of the formulations used to test the stability of ACE2 after spray freeze drying.

All the quantities are expressed in mg/ml except for ACE2, which is μg/ml.

Examples

Example 1: Morphological analysis

A generalized procedure for making spray freeze dried micro-particles to encapsulate protein active ingredient is shown in Figure 1A, whereby a formulation comprising a polymer, a carrier and an excipient are combined with an active agent (for example, a protein). The procedure shows freeze spraying of the formulation to produce an atomized droplet that is rapidly frozen in a cryogenic fluid, like liquid nitrogen (LN2), with a subsequent freeze-drying in a vacuum chamber to remove any residual moisture. Figure 1B shows the specific preferred apparatus set-up parameters for making spray freeze dried micro-particles to encapsulate proteins.

Analyzing the morphological properties of spray freeze dried micro-particle provide valuable insights on which formulations and methodologies produce a stable micro-particle or the desired size. For drug delivery, significant morphological properties include the average diameter of the micro-particles, the size distribution of the micro-particles, and the shape of the micro-particles. These parameters can be tuned and controlled by adjusting the total weight percentage, the ratio between polymer and carrier, the amino acid weight percentage, the ratio between two different carriers, and the overall makeup of the formulation being used for spray freeze drying. Figures 2, 3, 4, 5, show the effect of these parameters to the size distribution of spray freeze dried micro-particles. Furthermore, feeding flowrate of a) 15 ml/min and b) 5 ml/min are compared morphologically in Figure 8, while the effect of different freeze-drying procedure(i.e. a) 24 hours at 750 mBar; and b) 4 hours at 300 mTorr followed by 20 hours at 100 mTorr) are compared morphologically in Figure 9.

Similar morphologies are seen for spray freeze dried micro-particles comprising rhACE2 as shown in Figure 10. rhACE2 was mixed with PVP polymer, carrier and excipient at various concentrations and weight percentages (wt %), as shown in Table 1B. In a few formulations can be seen in Table 1B: 1) increase in amount of rhACE2, 2) change in total wt %, 3) difference in ratio between polymer and carrier (mannitol), 4) change in ratio between trileucine and leucine and trehalose and leucine, and a modification in one component. In the last group, the polymer used, PVP, is going to be used at different molecular weights, 55 and 360 KDa. Moreover, in this last group, leucine, the main amino acid used is substituted with arginine and tyrosine. The reason for selecting these conditions is to test which concentration could be considered optimal, meaning that at which concentration the size of the micro-particles is appropriate for nasal delivery and the rhACE2 is maintained stable. For micro-particles to be appropriate for nasal delivery, they would need to have an average projected equivalent diameter between about 100 μm and about 200 μm. As shown in Figure 10, almost every case of formulation that was used generate micro-particles with a size appropriate for nasal delivery. However, the use of lower molecular weights for PVP produces several "broken” micro-particles indicating that rhACE2 could be poorly encapsulated. In addition, diameters lower than 100 μm are reached when even a small percentage (1 wt %] of trehalose is added into the formulation. This result indicates that trehalose is not a good candidate for the purpose of producing encapsulated rhACE2 for nasal delivery.

Example 2: Protein encapsulation

In case of fluorescent proteins, a fluorescent confocal microscope can be used to image the position of the protein within the encapsulating shell, as shown in Figure 6. As shown, the bright and white areas, highlighted with a circle, indicate the presence of Bovine Serum Albumin (BSA] protein within the micro-particle.

While the confocal microscope provides a clear and visual validation of the location of the protein within the micro-particles, an alternative technique is required for a more accurate analysis. Error! Reference source not found, shows the typical peaks of oxygen, carbon, nitrogen, sulfur, and sodium of both reference materials and some of the samples shown in Error! Reference source not found.A obtained using X-ray photoelectron spectroscopy [XPS] . The atomic concentrations that are shown in Error! Reference source not found, relate to only the first 10 nm of the surface of spray freeze dried micro-particles. BSA shows a unique signature containing atomic concentration for S2p and Nals of 0.59 and 0.37, respectively. BSA is present in the first 10 nm of SFD micro-particles when these two types of atoms are detected.

A similar analysis is shown for rhACE2 in Figure 11, where encapsulated stable rhACE2 was produced as a potential micro-particle for reducing the infection rate of Covidl9 and similar airborne viruses.

On the morphology point of view, another aspect that could be analyzed is the distribution of the chemical components in the freeze dried micro-particles. For this purpose, two techniques have been used, a Confocal Scanning Microscope and an X-ray Photoelectron spectrometer. With the first, fluorescent rhACE2 was used in order to image the location at which the protein is within the spray freeze dried micro-particles. With the second, an analysis of the chemical compositions of the first 10 nm layers of spray freeze dried micro- particles can be achieved. In Figure 11, the outcomes of these two techniques are shown, at the top and at the bottom row, respectively. In these images, the data are obtained by using only one formulation containing, 6.0 mg/ml, 9.0 mg/ml, and 2.2 mg/ml, of PVP, Mannitol, and Leucine, respectively. For concentrations of rhACE2 lower than 20 μg/ml, the rhACE2 is distributed in between the center and the shell of spray freeze dried micro-particles. In addition, at the first 10 nm of freeze dried micro-particles, no signal from rhACE2 has been encountered with exception of the case containing a weight concentration of rhACE2 of 4.0 μg/ml. Example 3: Protein stability

The stability and activity of proteins are the most challenging characteristics to preserve while performing an encapsulation. BSA and the alternative proteins shown in Error! Reference source not found.A have a strong signal when hit by an infrared light. In particular, the IR peak located in the wavelength range 1600-1700 cm^-1 contains important references to the chemical structures of proteins. Figure 7 shows an example of deconvolution of this peak into four sub-peaks, β-sheet, α-helix, β-turn, and β-antiparallel. The IR spectra in the SI (not shown). In general, shift of the amine I peak (~1650 cm^-1) to the right could be caused by the interaction of nitrogen ions, present in the liquid nitrogen (LN2), with BSA, in particular with the C-O and C-N groups. This would occur in case of contact between BSA and liquid nitrogen and, thus, in case of an improper encapsulation. A lower and broader peak is a sign of protein denaturation. However, the percentage differs between the peaks of spray freeze dried encapsulated BSA and of free BSA. When a data point is compared to the reference free BSA, the closer data point to the reference, would show a null or minor degradation.

FTIR is considered a semi-quantitative technique. HPLC is known to show a higher accuracy and, in the case of proteins, it can be used to determine the quantity of protein contained in spray freeze dried micro-particles, the spraying yield, the change in the chemical structure of proteins, and the efficiency of encapsulation, as shown in Error! Reference source not found.. The calibration curves necessary to calculate the quantity of protein contained in spray freeze dried micro-particles are shown in Figures 12 and 13. The difference in area of the BSA monomer and the presence of dimers show a possible degradation of the protein.

The deconvolution curve and peak ratio are derived by analyzing the spectra shown in Figure 12. In this figure, only the wavelength range of 1600 and 1700 cnr¹ is shown since it relates to the amide I peak of BSA.

HPLC was also used to characterize micro-particles containing encapsulated BSA. Besides analyzing the encapsulation efficiency and stability of spray freeze drying, HPLC was used to quantify the BSA contained in the sprayed powder. To do so, a calibration curve is generated by analyzing aqueous samples containing a scaling range of BSA (0.33, 0.5, 0.75, 1, 1.25, 2.5, 5, and 10 mg/ml). Figure 13 shows two calibration curves, one derived by considering the peak area and one considering the peak height. Polynomials of 2^nd and 3^rd order are shown.

Another important property of these micro-particles is the stability of spray freeze dried encapsulated rhACE2. The technique used to analyze the stability of rhACE2 in spray freeze dried micro-particles are Fourier-transform infrared spectroscopy [FTIR], High- performance liquid chromatography (HPLC], Nuclear magnetic resonance (NMR], and ELISA. Results obtained using the FTIR are shown in Figure 12. In order to achieve a more quantitative analysis, the typical peaks of the Amide I of proteins has been deconvoluted. The smallest difference between the deconvoluted peak of spray freeze dried micro-particles and free rhACE2 is obtained for the formulations composed of 60, 90, 22 wt % of PVP, Mannitol, and Leucine, respectively and a wt% of rhACE2 lower than 20 μg/ml.

The result achieved using with the FTIR are supported and confirmed with the results achieved using the HPLC. Even though rhACE2 had not previously been analyzed with this technique, successful results were obtained, as shown in Figure 13.

In one case, NMR was used to support the results achieved with FTIR and HPLC. The case selected contains weight percentages (wt %] of 90, 60, and 22 for PVP, mannitol, and leucine, respectively, and 40 μg/ml of rhACE2. Figure 14 indicates, as seen in Figure 12 and Figure 13, that rhACE2 has undergone some structural changing while being spray freeze dried. However, the major peaks of free rhACE2 are still present in the spectrum of spray freeze dried micro-particles, indicating that these structural changes might not have damaged the activity of rhACE2.

The activity rate of encapsulated rhACE2 was tested using an ELISA kit on spray freeze dried micro-particles. Figure 15 shows ELISA analysis of spray freeze dried rhACE2 micro- particles, and show high encapsulation of rhACE2, whereby this method could be used for the production of high quantities of encapsulated rhACE2.

The second phase of the drying procedure, the freeze drying, can also influence the morphology and, thus, the properties of the powder. The selection of the temperature and vacuum pressure of the freeze-drying procedure could impact the physical and chemical properties of the dried micro-particles. Even though the relationship between the freeze- drying parameters and the properties of dried particles have been previously poorly analyzed [5], Figure 9 shows a clear example of this relationship. For the same sample, a vacuum pressure of 750 mTorr does not generate a powder composed of clear and separated spheres, which are an indication of a successful encapsulation.

Furthermore, the binding efficiency of encapsulated rhACE2 with the spike proteins of ACE2 was assessed (Figure 16 and Table 4). The binding efficiency did not increase with the increasing concentration of rhACE2 in the formulation. When the amount of rhACE2 was 0, 5, and 1 μg, the % of bioactive compounds was 89% and 77%, respectively. This percentage decreased with an increase in encapsulated rhACE2. Table 4 shows ratios of: leucine and mannitol; mannitol and PVP; trileucine and leucine; trehalose and leucine; of polymer with different molecular weights, with different amino acids.

Table 4. Percent Binding Efficiency

Example 4: Total solids weight

In spray drying techniques, it is well-known that the total solids weight percentage (tw) is proportional to the projected area equivalent diameter (da) in spray dried powders [34]. For a ratio between polymer and carrier [r] of 1.2, increasing the tw from 14.6 to 22.1 mg/ml generates a surge in da from 110 to 220 μm, Figure 2a. By dividing r by a half, higher tw appears to stimulate an increase in diameter; a difference in tw of 16.5 mg/ml produces an increase in da of 120 μm. A richer solution would create more viscous droplets, which tend to produce spray-dried micro-particles with a larger diameter [72]. However, the two-flow nozzle shows a limitation in viscosity, which is reflected in the highest tw with an r of 1.2. In these cases, the micro-particles are not formed.

PVP shows an average solubility of 100 mg/ml. By employing agitation, the solubility could be further increased, boosting the solution’s viscosity and reducing the ability of other components to dissolve [72]. The poor encapsulation efficiency reflects the lack of particle formation for the cases of high tw and r of 1.2. The gap between the peak areas of [ β-sheet and β-antiparallel of the powder obtained in these cases and pure BSA reaches the value of 150, as shown in Figure 7a. In addition, the mass peak area of the dimer 1297 for r 1.2 and 1432 for r ¼ 0.6 for tw of 29.1 and 39.6 mg/ml, respectively. As seen in the SEM images in Figure 3a, micro-particles are not formed and the BSA is not homogenously spread in the powder, making the exposed BSA prone to damage. In fact, BSA formed dimers; the mass peak area of the dimer 1297 for r 1.2 and 1432 for r ¼ 0.6 for tw of 29.1 and 39.6 mg/ml respectively. The dimer area disappears when the tw goes below 19 mg/ml, showing also one of the highest encapsulation efficiencies [g] [90.8], Table 3. The encapsulation for these cases is confirmed by the difference between the area of the monomer in mass spectrum of the spray freeze dried micro-particles and pure BSA [< 1.05 %, Table 3]. For one of these cases, BSA is confirmed to be positioned within the micro-particle’s shell, Figure 6.

Example 5: Ratio between polymer and carrier

Due to the promising results achieved by employing a tw below 19, a tw of 17.1 is selected to verify the impact of r to the properties of SFD micro-particles. As described in the above sub-sections, the cases containing PVP at weights above 10 mg/ml do not or only partially generate micro-particles, Figure 2a. Such a conclusion has been shown by Sou et aL, who showed that weight percentage ratios of 60 and above of PVP enhance the protein stability of spray-dried powder [73]. For r below than 5, a decrease in r contributes to a decrease in da by maintaining the BSA located inside the micro-particle shell, Figure 6b and c. A reduction in r is achieved by reducing PVP and raising the amount of carrier, mannitol, or lactose. PVP distributes first on the surface of the evaporating droplet; the time to shell formation could be delayed (even by a few milliseconds) when having a lesser number of PVP molecules [34]. A delayed time to shell formation would imply micro-particles with a slightly smaller diameter. An opposite trend is shown in Figure 2a underlining the importance of the carrier. As demonstrated by Eggerstedt et al., lower values of r produced micro-particles with a surface composed of a small number of concentric patterns and an inner core highly porous [5]. This might indicate that when the droplet reaches the nitrogen, the shell is not fully formed. The freeze-drying procedure removes the remaining water. Due to the large molecular weight and the large content in the initial formulation of PVP 1300, the shell formation is expected to occur in a few milliseconds at temperatures below 20 °C [34, 36, 44]. The shell formation at 10 cm distance can be verified by the fact that when increasing the speed of liquid flow, no micro-particles are formed (not shown]. Therefore, it can be assumed that the first layer of the shell is formed before reaching the liquid nitrogen container. The porous structure is then derived by the water leaving the formed shell during the freeze-drying. This assumption might be valid for most cases shown under this category. A larger da for lower r could be related to the effect of the remaining water in SFD micro- particles. Some coagulation is visible in images of micro-particles taken for r of 0.6 for both mannitol and lactose as carriers, Figure 2a. The coagulated appearance is much more visible for all cases involving lactose as the sole carrier. Lactose has a higher tendency to crystallize under a certain pressure, at low levels of humidity, or exposed to high temperatures [9]. This fragility of lactose could have damaged the particles’ structure during the freeze drying procedure where remaining water is removed at different vacuum pressures and temperatures. For the cases containing lactose solely, the difference between the IR peaks β-sheet and β-turn is, on average, 50% higher than the corresponding mannitol-containing cases, Figure 7b. The lowest g [63.4] is connected to the case with a r of 2 and contains lactose, Table 3. High dimer areas in the mass spectra of cases containing lactose and with high r are shown in Table 3. The exact opposite result is obtained analyzing samples containing mannitol and with a low r (0.6 and 0.3), Table 3. This further remarks that mannitol is an optimal carrier for the stability of drugs [33].

Example 6: Ratio between amino acid and carrier

Leucine has an impressive effect on the particle formation generated by spray drying techniques [11]. L-leucine has a high Peclet number [Pe] and, thus, during the drying procedure, it precipitates on the surface of droplets producing a hydrophobic layer. This layer restricts the dispersion of water and brings the formation of indented particles [74]. Sou et al.. identify the weight percentage of 20 as the optimal for maintaining the protein stability in spray drying formulations containing PVP and mannitol [73]. Therefore, values of L-leucine weights between 0 and 4.5 have been tested to understand the effect on BSA stability. Without any leucine, at a ratio of 0 between the amino acid and the carrier [ra], the particles look highly porous and the surface highly cracked. Therefore, the stability of BSA can be compromised. All the b-peak areas of this case differ from the pure BSA of an average of 100, Figure 3a. The difference between the monomer area with pure BSA is also reasonably high, 176, Table 3. By increasing the weight of leucine (up to reaching a ra of 0.15], the SFD micro-particles appear smoother and with a lower content of pores in their internal structure, Figure 3a.

Moreover, by analyzing fluorescent images, the BSA is ensured to be enclosed, Figure 6, by a layer of L-leucine and PVP (first 10 nm of the micro-particles as listed in Table 2]. Weights of L-leucine between 1.5 and 2.5 seem to be ideal for maintaining the BSA stability. From the IR and chromatography spectra analysis, none of the significant peaks show a change compared to free BSA, as shown in Figure 7c and Table 3. Beyond 2.5 mg/ml, L-leucine promotes a very early shell formation, producing larger micro-particles; with an increase in ra from 0.2 to 1, the da raises from 180 to 340 μm. However, the shell formed tends to be weak, and, once frozen, a larger quantity of water is entrapped within. The reasonably quick freeze-drying procedure could provoke the rupture of such a weak shell and the formation of a highly porous internal structure. These two characteristics make these micro-particles very similar in morphology to the case of 0 mg/ml of L-leucine, Figure 7c. Example 7: Ratio between two carriers

A co-spray drying of carriers, such as lactose and mannitol, is often used [12]. For example, Ferdynand and Nockhodchi show a ratio of 1:3 mannitoldactose ratio to offer the highest salbutamol sulfate stability in spray-dried powders [12]. Here, we try to investigate the conjunct action of two carriers to the particle formation of SFD micro-particles, including one or more proteins. With an increase in the ratio between mannitol and lactose [rc], the particles show a higher da and a smoother surface [Figure 3b). The larger diameter is due to the presence of mannitol and with a high Peclet number, it distributes quicker to the surface of the evaporating droplet. The rougher surface in cases of high lactose content is derived by, possibly, the late time to shell formation. If a thin shell is formed by the time evaporating droplets are collected into the liquid nitrogen, the freeze-drying procedure might break or crack it while the water is leaving. Even though the BSA appears to be enclosed in all cases tested under this category, Figure 3b shows that BSA’s stability is spoiled by high lactose content. By decreasing rc from 8 to 0.1, the dimer area increases from 64 to 1133, the difference between the monomer area of spray-dried micro-particles and pure BSA increases from 34 to 134, Table 3, and the IR peaks of [β-sheet and [β-antiparallel deviate from the pure BSA ones of more than 150 %, Figure 7. A high encapsulation efficiency, 71.3, is reached for rc of 8. Again the cause of this success can be linked to the early shell formation supported by mannitol [75] and to the possible higher tendency of lactose to crystallize [76].

Example 8: Components

Protein to encapsulate

Encapsulating proteins with a broad range of molecular weight indicates the efficiency of the formulation developed. We select the weight percentages of 60, 90, and 20 for PVP, Mannitol, and Leucine, respectively due to the BSA stability at these conditions [Figure 6, Figure 7, and Error! Reference source not found.). As shown in Figure 4a, the morphology and the d_a of freeze spray dry micro-particles are not altered by a change in protein. The micro and nano roughness appear unmodified with different types of proteins Figure 4a. However, a correlation between the micro-particles with largest size and the degradation rate seems proportional. In fact, micro-particles composed by whey, pea, and soy protein show a d_a close or larger than 200 μm, Figure 4a. The same cases show a difference between the deconvoluted β-sheet of the freeze dried micro-particles and free BSA of about 100, 20, and 20 respectively, Figure 7h. Besides the molecular weight, there are other differences between the proteins selected. First of all, β-lactoglobulin and hemoglobulin have a chemical composition much shorter compare to pea, whey, and soy proteins. The last contain more than 20 components, which could contribute to a facile degradation during the processes of spray drying in a cryogenic liquid, and of freeze drying. Even so, dimers were low in number or not present in the mass spectrum of micro-particles containing different types of proteins, Error! Reference source not found.. A mild decrease of the stability can be seen in the difference in the monomer area for micro-particles containing whey and soy protein, 86.2 and 133 respectively, Error! Reference source not found..

Polymer for shell formation

PVP can be available at different weight percentages. High molecular weight polymers tend to distribute earlier on the surface of an evaporating droplet and may explain why the use of PVP with a molecular weight of 1300 kDa generates spherical and smooth micro-particles, Figure 4b. Using the same polymer with a lower molecular weight should produce a late shell formation and, thus, possibly larger micro-particles in a common spray drying procedure. However, when spray drying in a cryogenic fluid using PVP with a molecular weight of either 360 kDa and 55 kDa, the shell seems not to be fully formed at the moment of reaching the liquid nitrogen. This assumption is confirmed by the large and agglomerated particles visualized in the SEM images, Figure 4b. Not completing the shell formation would obviously result in a sharp decrease of BSA stability, as shown in the IR and mass spectra peaks deconvolution in Figure 7f and Error! Reference source not found.. Although PVP 360 showed good encapsulation efficiency, there is a significant reduction in drug stability of the micro-particles as compared to PVP 1300 as shown in the FTIR curves and by the HPLC data (there is more gap between the pure and encapsulated drug). Therefore, even though PVP 1300 has a slightly lower in encapsulation efficiency, it has a higher drug stability and more appropriately sized micro-particles than PVP 360. Type of amino acid

Leucine has been used in spray drying due to its known quality in improving the aerodynamic properties of the particles aimed for some types of drug delivery [i.e. pulmonary) [11]. Moreover, hydrophobic amino acids are able to protect spray dried formulations against moisture changes. Trileucine is considered a surface-active molecule and is shown to improve dispersibility of particles without altering the morphology of the particle [36]. However, a high amount of trileucine might generate a coating on the surface and increase the cohesiveness of spray dried micro-particles [36]. The rate of coagulated particles and, thus, the number of particles with high cohesiveness, appears to increase proportionally to a surge of the ratio between trileucine and leucine [Figure 5a]. As a result, the average d_a increases from 178 to about 400 μm with an increase in the ratio of trileucine and leucine from 0 to 3, as shown in Figure 5a. With the same increase in ratio, the dimer area of the mass spectra increases from 0 to 890 mAU*s, Error! Reference source not found..

Amino acids can be divided into charged, polar, amphipathic, and hydrophobic. Arginine falls in the first category, where members, having side chains, can form salt bridges. As solution additive, arginine is used to stabilize proteins against aggregation, especially in the process of protein refolding [77]. Moreover, when distributed on the surface of spray dried micro- particles, arginine has been reported to enhance the dispersibility due to the positive charge and generates electrostatic repulsion between particles [77]. As shown in Figure 5b, the separation between micro-particles containing arginine is sharper compared to micro- particles containing other amino acids. This morphological property could be beneficial for some types of drug delivery, i.e., aggregation affects the location of deposition in nasal or lung delivery [43]. Arginine appears to maintain the stability of proteins as shown with a maximum distance in the deconvoluted IR peaks with respect to free BSA of only 2 %, Figure 7g.

Glutamine is polar and tends to form hydrogen bonds as proton donor. However, glutamine is shown to have a minimal effect on the protein stability [78]. In fact, even though changes on the morphology compared to micro-particles containing other amino acids is practically null Figure 5b, a slight degradation on the BSA stability can be recognized. For example, the difference of all IR peaks of spray freeze dried micro-particles and free BSA is almost 10 % and a minor presence of a dimer area of 5.5 mAU*s in the IR spectra, Error! Reference source not found..

Tyrosine belongs to the group of amphipathic amino acids since it can show both a polar and non-polar behavior [79]. The oxygen bonds generated by -OH groups of tyrosine are known to highly contribute to the stability of proteins [36]. This amino acid is much less common to be used in spray drying since it does not contribute directly to the shell or particle formation. However, given enough time for the distribution of the other components on the surface of evaporating droplets, the use of tyrosine highly contributes to the stability of proteins. Micro-particles containing tyrosine shows the lowest difference in monomer area respect with free BSA [1.23 in Error! Reference source not found.]. Moreover, all the IR peaks differ at the most of 4 % from the free BSA peaks, Figure 7g.

Alternative polymers

The choice of polymer and the particular molecular weight [M_w] of the polymer may be guided by a wide variety of factors, each of which may be weighted differently depending on the other characteristics associated with the polymer. A polymer having very low diffusivity, which is the speed at which the molecules travel from the surface to the center of the evaporating droplet is important. Accordingly, a low diffusivity is connected to a high Peclet number, which is the ratio between the evaporation rate of the solvent, and the diffusivity of the solute in the selected solvent. Polymers having a high Peclet number are usually associated with large spray dried micro-particles because the time for shell formation is short. This short time for the first layer of the shell to be formed allows for shell formation fairly close to the spraying atomizer. This is significant for spray freeze drying, since the slurry droplets are collected into a container of liquid nitrogen positioned only 10 cm below the spraying atomizer. If a proper shell is not formed before the slurry reaches the liquid nitrogen, a proper encapsulation will not be achieved. Furthermore, the thicker the shell is at the collection point and the stronger the shell can withstand the pressure of the water evaporating through its pore during the phase of freeze drying. However, other factors are also applicable in determining the characteristics of encapsulating micro-particles are: molecular weight; solubility; viscosity; and toxicity. Table 5 shows a comparison of a sampling of polymers showing the wide range of characteristics associated with each polymer and further with each M_n polymer. The choice of polymer greatly depends on many factors associated with the polymer itself and the carrier(s) and/or excipient(s) the polymer is to be combined with and the ultimate use for the encapsulated micro-particles.

Table 5. Polymer Comparisons

Further testing with alternative polymers (i.e. soluble cellulose at 570 kDa and chitosan at 1500 kDa, with 9 mg/ml mannitol and 2 mg/ml of leucine, plus protein) did not form useful powders. Although, PVP and chitosan are hydrophobic, chitosan is less soluble in water compared to PVP. Furthermore, the cellulose tested had a lower M_n compared to the PVP used and cellulose is hydrophilic. Similar to cellulose, gelatin is also hydrophilic. Peclet numbers cannot be defined easily as it will depend on the quantities in water and on the diffusivity of these materials in water, with a dependence on temperature.

Conclusions

An increase in total solids weight generates a corresponding increase in the projected equivalent area of micro-particles and can decrease the stability of encapsulated proteins, despite of the ratio between polymer and carrier. High quantities of amino acid contained in the formulation can produce an increase in the projected equivalent area of micro- particles. Weights between 1.5 to 2.5 mg/ml show the lowest damage to the encapsulated proteins. Mannitol is preferred over lactose in preserving the stability of encapsulated proteins. The formulation composed of PVP, mannitol, and leucine at the weights of 6, 9, and 2 mg/ml, respectively, is a preferred formulation to encapsulate stable proteins of several types. The polymer PVP preferably has a molecular weight of 1300 kDa for ensuring its distribution on the surface of micro-particles. While the morphology remains intact when changing the kind of amino acid, the stability can be reduced when glutamine is the amino acid excipient.

The disclosure may be further understood by the non-limiting examples. Although the description herein contains many specific examples, these should not be construed as limiting the scope of the disclosure but as merely providing illustrations of some of the embodiments of the disclosure. For example, thus the scope of the disclosure should be determined by the appended aspects and their equivalents, rather than by the examples given.

Many of the molecules disclosed herein may contain one or more ionizable groups [groups from which a proton can be removed [e.g., -COOH) or added [e.g., amines) or which can be quaternized [e.g., amines)]. Where appropriate all possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art can select from among a wide variety of available counter-ions those that are appropriate for preparation of salts of this disclosure for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt may result in increased or decreased solubility of that salt. Every formulation or combination of components described or exemplified herein may be used to practice the disclosure, unless otherwise stated.

Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising” is used herein as an open ended term, substantially equivalent to the phrase "including, but not limited to”, and the word "comprises” has a corresponding meaning. As used herein, the singular forms "a”, "an” and "the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed disclosure belongs. References

[1] S.D. Putney, Encapsulation of proteins for improved delivery, Current Opinion in Chemical Biology, 2 (1998) 548-552.

[2] C. Ye, H. Chi, A review of recent progress in drug and protein encapsulation: Approaches, applications and challenges, Materials Science and Engineering: C, 83 (2018) 233-246.

[3] L. Wang, Y. Liu, W. Zhang, X. Chen, T. Yang, G. Ma, Microspheres and microcapsules for protein delivery: strategies of drug activity retention, Current Pharmaceutical Design, 19 (2013) 6340-6352.

[4] Y.-F. Maa, P.-A. Nguyen, T. Sweeney, S.J. Shire, C.C. Hsu, Protein inhalation powders: spray drying vs spray freeze drying, Pharmaceutical Research, 16 (1999) 249-254.

[5] S.N. Eggerstedt, M. Dietzel, M. Sommerfeld, R. Siiverkrup, A. Lamprecht, Protein spheres prepared by drop jet freeze drying, International Journal of Pharmaceutics, 438 (2012) 160- 166.

[6] T.L. Rogers, J. Hu, Z. Yu, K.P. Johnston, R.O. Williams HI, A novel particle engineering technology: spray-freezing into liquid, International Journal of Pharmaceutics, 242 (2002) 93-100.

[7] C. Cavallari, B. Luppi, A.M. Di Pietra, L. Rodriguez, A. Fini, Enhanced release of indomethacin from PVP/stearic acid microcapsules prepared coupling co-freeze-drying and ultrasound assisted spray-congealing process, Pharmaceutical Research, 24 (2007) 521-529.

[8] H.R. Costantino, L. Firouzabadian, C. Wu, K.G. Carrasquillo, K. Griebenow, S.E. Zale, M.A. Tracy, Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability, Journal of Pharmaceutical Sciences, 91 (2002) 388-395.

[9] L. Wu, X. Miao, Z. Shan, Y. Huang, L. Li, X. Pan, Q. Yao, G. Li, C. Wu, Studies on the spray dried lactose as carrier for dry powder inhalation, Asian Journal of Pharmaceutical Sciences, 9 (2014) 336-341. [10] W.L. Hulse, R.T. Forbes, M.C. Bonner, M. Getrost, The characterization and comparison of spray-dried mannitol samples, Drug Develoμment and Industrial Pharmacy, 35 [2009] 712-718.

[11] M.Y. Chow, Y. Qiu, F.F. Lo, H.H. Lin, H.-K. Chan, P.C. Kwok, J.K. Lam, Inhaled powder formulation of naked siRNA using spray drying technology with L-leucine as dispersion enhancer, International Journal of Pharmaceutics, 530 [2017] 40-52.

[12] M.S. Ferdynand, A. Nokhodchi, Co-spraying of carriers [mannitol-lactose] as a method to improve aerosolization performance of salbutamol sulfate dry powder inhaler, Drug Delivery and Translational Research, [2020] 1-10.

[13] T. M. Serim, J. Kozak, A. Rautenberg, A. N. Ozdemir, Y. Pellequer, A. Lamprecht, Spray Freeze Dried Lyospheres for Nasal Administration of Insulin, Pharmaceutics [2021] 13[6]:852.

[14] GW Oetjen, P Haseley Freeze-drying 2^nd edition [2004] Wiley - VCH.

[15] Lippi G, Mattiuzzi C, Henry BM. Updated picture of SARS-CoV-2 variants and mutations. Diagnosis 9, 11-17 [2022).

[16] Rabaan AA et al.. Airborne transmission of SARS-CoV-2 is the dominant route of transmission: droplets and aerosols. Infez Med 29, 10-19 [2021].

[17] Tang JW, et al. Dismantling myths on the airborne transmission of severe acute respiratory syndrome coronavirus [SARS-CoV-2]. Journal of Hospital Infection, [2021].

[18] Greenhalgh T, Jimenez JL, Prather KA, Tufekci Z, Fisman D, Schooley R. Ten scientific reasons in support of airborne transmission of SARS-CoV-2. The Lancet 397, 1603-1605 [2021],

[19] McNeill VF. Airborne Transmission of SARS-CoV-2: Evidence and Implications for Engineering Controls. Annual Review of Chemical and Biomolecular Engineering 13, [2022]. [20] Cheng YS. Aerosol deposition in the extrathoracic region. Aerosol Science & Technology 37, 659-671 (2003],

[21] Klingenstein M, et al. Evidence ofSARS-CoV2 entry protein ACE2 in the human nose and olfactory bulb. Cells Tissues Organs 209, 155-164 [2020].

[22] Xiao L, Sakagami H, Miwa N. ACE2: the key molecule for understanding the pathophysiology of severe and critical conditions of COVID-19: demon or angel? Viruses 12, 491 (2020],

[23] Zeinalian M, Salari-Jazi A, Jannesari A, Khanahmad H. A potential protective role of losartan against coronavirus-induced lung damage. Infection Control & Hospital Epidemiology 41, 752-753 [2020],

[24] Furuhashi M, Moniwa N, Takizawa H, Ura N, Shimamoto K. Potential differential effects of renin-angiotensin system inhibitors on SARS-CoV-2 infection and lung injury in COVID- 19. Hypertension Research, 1-4 [2020],

[25] Gurwitz D. Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics. Drug Development Research, [2020],

[26] Rossi GP, Sanga V, Barton M. Potential harmful effects of discontinuing ACE-inhibitors and ARBs in COVID-19 patients. Elife 9, e57278 [2020],

[27] Pang X, Cui Y, Zhu Y. Recombinant human ACE2: potential therapeutics of SARS-CoV-2 infection and its complication. Acta Pharmacologica Sinica 41, 1255-1257 [2020],

[28] Monteil V, et al.. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell, [2020],

[29] Alhenc-Gelas F, Drueke TB. Blockade of SARS-CoV-2 infection by recombinant soluble ACE2. Kidney International, [2020],

[30] Bathe D, Wysocki J, Satchell K. Soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy? Clinical Science 134, 543-545 [2020], [31] Zhang H, Penninger JM, Li Y, Zhong N, Slutsky AS. Angiotensin-converting enzyme 2 [ACE2] as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Medicine, 1-5 [2020].

[32] Wadman M, Couzin-Frankel J, Kaiser J, Matacic C. How does coronavirus kill. Clinicians Trace a Ferocious Rampage Through the Body, From Brain to Toes, 1502-1503 [2020].

[33] Shetty N, Cipolla D, Park H, Zhou QT. Physical stability of dry powder inhaler formulations. Expert Opinion on Drug Delivery 17, 77-96 [2020].

[34] Baldelli A, Boraey MA, Nobes DS, Vehring R. Analysis of the particle formation process of structured micro-particles. Molecular pharmaceutics 12, 2562-2573 [2015].

[35] Wysocki J, Schulze A, Bathe D. Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the Kidney Renin Angiotensin System. Biomolecules 9, 886 [2019],

[36] Vehring R. Pharmaceutical particle engineering via spray drying. Pharmaceutical Research 25, 999-1022 [2008],

[37] Sangolkar S, Adhao V, Mundhe D, Sawarkar H. Particle size determination of nasal drug delivery system: A review. IntJ Pharm Sci Rev Res 17, 66-73 [2012].

[38] Arora P, Sharma S, Garg S. Permeability issues in nasal drug delivery. Drug Discovery Today 7, 967-975 [2002],

[39] Augustin M, Sanguansri L. Encapsulation of bioactives. In: Food Materials Science). Springer [2008].

[40] Akbarbaglu Z, Peighambardoust SH, Sarabandi K, Jafari SM. Spray drying encapsulation of bioactive compounds within protein-based carriers; different options and applications. Food Chemistry, 129965 [2021].

[41] Onwulata C. Encapsulation of new active ingredients. Annual Review of Food Science and Technology 3, 183-202 [2012]. [42] Maruyama T, et al.. FT-IR analysis of BSA fouled on ultrafiltration and microfiltration membranes. Journal of Membrane Science 192, 201-207 [2001].

[43] Baldelli A, Vehring R. Control of the radial distribution of chemical components in spray- dried crystalline micro-particles. Aerosol Science and Technology 50, 1130-1142 [2016].

[44] Baldelli A, Power RM, Miles RE, Reid JP, Vehring R. Effect of crystallization kinetics on the properties of spray dried micro-particles. Aerosol Science and Technology 50, 693-704 [2016],

[45] Horvath T, et al.. Cytotoxicity of Different Excipients on RPMI 2650 Human Nasal Epithelial Cells. Molecules [Basel, Switzerland) 21, 658 [2016].

[46] Reichl S, Becker K. Cultivation of RPMI 2650 cells as an in-vitro model for human transmucosal nasal drug absorption studies: optimization of selected culture conditions. Journal of Pharmacy and Pharmacology 64, 1621-1630 [2012].

[47] Haghi M, et al. Immunomodulatory effects of a low-dose clarithromycin-based macrolide solution pressurised metered dose inhaler. Pharmaceutical Research 32, 2144- 2153 [2015],

[48] Grau-Bartual S, Al-Jumaily AM, Young PM, Traini D, Ghadiri M. Effect of continuous positive airway pressure treatment on permeability, inflammation and mucus production of human epithelial cells. ERJ Open Research 6, 00327-02019 [2020].

[49] Hoekman JD, Ho RJY. Effects of localized hydrophilic mannitol and hydrophobic nelfinavir administration targeted to olfactory epithelium on brain distribution. Aaps Pharmscitech 12, 534-543 [2011].

[50] Giuliani A et al.. In vivo nose-to-brain delivery of the hydrophilic antiviral ribavirin by micro-particle agglomerates. Drug Delivery 25, 376-387 [2018].

[51] Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems./ LabAutom 20, 107-126 [2015]. [52] Pozzoli M, et al. Application of RPMI 2650 nasal cell model to a 3D printed apparatus for the testing of drug deposition and permeation of nasal products. EurJ Pharm Biopharm 107, 223-233 [2016],

[53] Bai S, Yang T, Abbruscato TJ, Ahsan F. Evaluation of human nasal RPMI 2650 cells grown at an air-liquid interface as a model for nasal drug transport studies.J Pharm Sci-Us 97, 1165- 1178 [2008],

[54] Laksitorini M, Prasasty VD, Kiptoo PK, Siahaan TJ. Pathways and progress in improving drug delivery through the intestinal mucosa and blood-brain barriers. Therapeutic Delivery 5, 1143-1163 (2014).

[55] Littringer EM, et al.. Spray drying of mannitol as a drug carrier — the impact of process parameters on product properties. Drying Technology 30, 114-124 (2012).

[56] Ordoubadi M, et al.. On the particle formation of leucine in spray drying of inhalable micro-particles. International Journal of Pharmaceutics 592, 120102 (2021).

[57] Sayani AP, Chun IK, Chien YW. Transmucosal delivery of leucine enkephalin: stabilization in rabbit enzyme extracts and enhancement of permeation through mucosae. Journal of Pharmaceutical Sciences 82, 1179-1185 (1993).

[58] Holbrook PA. Biological barriers to the nasal delivery of peptide drugs.). Aston University (1991).

[59] Baldelli A, Vehring R. Analysis of cohesion forces between monodisperse micro- particles with rough surfaces. Colloids and Surfaces A: Physicochemical and Engineering Aspects 506, 179-189 (2016).

[60] Wang Y, Kho K, Cheow WS, Hadinoto K. A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid-polymer hybrid nanoparticles. International Journal of Pharmaceutics 424, 98-106 (2012). [61] Poozesh S, et al.. Understanding the process-product-performance interplay of spray dried drug-polymer systems through complete structural and chemical characterization of single spray dried particles. Powder Technology 320, 685-695 [2017].

[62] Costantino HR, et al.. Protein spray-freeze drying. Effect of atomization conditions on particle size and stability. Pharmaceutical Research 17, 1374-1382 [2000].

[63] Carrasquillo KG, et al.. Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly [lactide-co-glycolide] microspheres results in release of native protein. Journal of Controlled Release 76, 199-208 [2001].

[64] Oudit GY, et al.. Human recombinant ACE2 reduces the progression of diabetic nephropathy. Diabetes 59, 529-538 [2010].

[65] Lu R, Li W-W, Katzir A, Raichlin Y, Yu H-Q, Mizaikoff B. Probing the secondary structure of bovine serum albumin during heat-induced denaturation using mid-infrared fiberoptic sensors. Analyst 140, 765-770 [2015].

[66] Haque MA, Adhikari B. Drying and denaturation of proteins in spray drying process. Handbook of Industrial Drying, 971-985 [2015].

[67] Lam XM, Duenas ET, Daugherty AL, Levin N, Cleland JL. Sustained release of recombinant human insulin-like growth factor-I for treatment of diabetes. Journal of Controlled Release 67, 281-292 [2000].

[68] Walker SN, et al. SARS-CoV-2 assays to detect functional antibody responses that block ACE2 recognition in vaccinated animals and infected patients. Journal of Clinical Microbiology 58, [2020],

[69] Fallacara A et al. In vitro characterization of physico-chemical properties, cytotoxicity, bioactivity of urea-crosslinked hyaluronic acid and sodium ascorbyl phosphate nasal powder formulation. International Journal of Pharmaceutics 558, 341-350 [2019].

[70] Ong HX, et al.. Primary Air-Liquid Interface Culture of Nasal Epithelium for Nasal Drug Delivery. Mol Pharmaceut 13, 2242-2252 [2016]. [71] Guidance D. Bioavailability and bioequivalence studies for nasal aerosols and nasal sprays for local action. Center for Drug Evaluation and Research, Rockville, MD [2003].

[72] Desai, K. G. H.; Park, H. J. Preparation and Characterization of Drug-Loaded Chitosan- Tripolyphosphate Microspheres by Spray Drying. Drug Dev. Res. 2005, 64, 114-128.

[73] Sou, T.; McIntosh, M. P.; Kaminskas, L. M.; Prankerd, R. ].; Morton, D. A. Designing a Multicomponent Spray-Dried Formulation Platform for Pulmonary Delivery of Biomacromolecules: The Effect of Polymers on the Formation of an Amorphous Matrix for Glassy State Stabilization of Biomacromolecules. Drying Technol. 2013, 31, 1451-1458.

[74] Lechanteur, A.; Evrard, B. Influence of Composition and Spray-Drying Process Parameters on Carrier-Free DPI Properties and Behaviors in the Lung: A Review. Pharmaceutics. 2020, 12, 55.

[75] Maas, S. G.; Schaldach, G.; Littringer, E. M.; Mescher, A.; Griesser, U. ].; Braun, D. E.; Walzel,P. E.; Urbanetz, N. A. The Impact of Spray Drying Outlet Temperature on the Particle Morphology of Mannitol. Powder Technol. 2011, 213,27-35.

[76] Haque, M. K.; Roos, Y. H. Crystallization and X-Ray Diffraction of Spray-Dried and Freeze- Dried Amorphous Lactose. Carbohydr. Res. 2005, 340,293-301.

[77] Lu, W.; Rades, T.; Rantanen, ].; Yang, M. Inhalable co-Amorphous Budesonide-Arginine Dry Powders Prepared by Spray Drying. Int. J. Pharm. 2019, 565, 1-8.

[78] Ladurner, A. G.; Fersht, A. R. Glutamine, Alanine or Glycine Repeats Inserted into the Loop of a Protein Have Minimal Effects on Stability and Folding Rates. J. Mol. Biol. 1997, 273, 330-337.

[79] Scheiner, S.; Kar, T.; Pattanayak, J. Comparison of Various Types of Hydrogen Bonds Involving Aromatic Amino Acids. J. Am. Chem. Soc. 2002, 124, 13257-13264.

Claims

Claims

1. A formulation for spray freeze drying, the formulation for spray freeze drying comprising:

(a) a polyvinyl pyrrolidone (PVP) polymer having an average molecular weight of at least 1,300 kDa;

(b) one or more sugars or sugar alcohols;

(c) one or more amino acids; and

(d) an active ingredient.

2. The formulation of claim 1, wherein the active ingredient is a protein or a fragment thereof.

3. The formulation of claim 1 or 2, wherein the active ingredient is a protein or a fragment thereof at a concentration up to 10μg/ml.

4. The formulation of claim 1, 2, or 3, wherein the active ingredient is an angiotensin converting enzyme 2 (ACE2) or a fragment thereof.

5. The formulation of any one of claims 1-4, wherein the one or more sugars or sugar alcohols are selected from one or more of: lactose; glucose; sucrose; mannitol; xylitol; sorbitol; and trehalose.

6. The formulation of any one of claims 1-5, wherein the one or more sugars or sugar alcohols is mannitol and has a concentration between 9-11 mg/ml.

7. The formulation of any one of claims 1-5, wherein one or more sugars or sugar alcohols are mannitol and lactose.

8. The formulation of claim 7, wherein the ratio of mannitol:lactose is 8:1.

9. The formulation of any one of claims 1-8, wherein the one or more amino acids is a hydrophobic amino acid.

10. The formulation of any one of claims 1-9, wherein the one or more amino acids is selected from one or more of: leucine; tyrosine; alanine; isoleucine; lysine; histidine; threonine; cysteine; methionine; phenylalanine; tryptophan; and valine.

11. The formulation of any one of claims 1-8, wherein the one or more amino acids is selected from one or more of: leucine; tri-leucine; arginine; glutamine; and tyrosine.

12. The formulation of any one of claims 1-11, wherein the one or more amino acids is leucine and has a concentration between 2-2.2 mg/ml.

13. The formulation of any one of claims 1-11, wherein the one or more amino acids is within the weight percentage range of 15% - 25%.

14. The formulation of any one of claims 1-13, wherein the PVP has a concentration between 6-7 mg/ml.

15. The formulation of any one of claims 1-13, wherein the PVP has a concentration of 6 mg/ml.

16. A micro-particle composition, the micro-particle composition comprising:

(a) a PVP polymer having an average molecular weight of at least 1,300 kDa;

(b) one or more sugars or sugar alcohols;

(c) one or more amino acids; and

(d) an active ingredient.

17. The micro-particle composition of claim 16, wherein the micro-particle has a diameter between 100-200 μm.

18. The micro-particle composition of claims 16 or 17, wherein the micro-particle has a projected area equivalent diameter [d_a] of about 100 μm to about 200 μm.

19. The micro-particle composition of claim 16, 17 or 18, wherein the active ingredient is a protein or a fragment thereof.

20. The micro-particle composition of any one of claims 16-19, wherein the active ingredient is an ACE2 protein or a fragment thereof.

21. The micro-particle composition of any one of claims 16-20, wherein the active ingredient is a protein or a fragment thereof is present at up to 10μg per micro-particle.

22. The micro-particle composition of any one of claims 16-21, wherein the micro- particle is suitable for nasal administration.

23. The micro-particle composition of any one of claims 16-22, wherein the one or more sugars or sugar alcohols are selected from one or more of: lactose; glucose; sucrose; mannitol; xylitol; sorbitol; and trehalose.

24. The micro-particle composition of any one of claims 16-23, wherein the one or more sugars or sugar alcohols is mannitol and is between about 49.45 % and about 57.89 % by weight of the final composition.

25. The micro-particle composition of any one of claims 16-23, wherein one or more sugars or sugar alcohols are mannitol and lactose.

26. The micro-particle composition of claim 25, wherein the ratio of mannitoldactose is 8:1.

27. The micro-particle composition of any one of claims 16-26, wherein the one or more amino acids is a hydrophobic amino acid.

28. The micro-particle composition of any one of claims 16-27, wherein the one or more amino acids is selected from one or more of: leucine; tyrosine; alanine; isoleucine; lysine; histidine; threonine; cysteine; methionine; phenylalanine; tryptophan; and valine.

29. The micro-particle composition of any one of claims 16-28, wherein the one or more amino acids is selected from one or more of: leucine; tri-leucine; arginine; glutamine; and tyrosine.

30. The micro-particle composition of any one of claims 16-29, wherein the one or more amino acids is leucine and is between about 10.00 % and about 12.79 % by weight of the final composition.

31. The micro-particle composition of any one of claims 16-29, wherein the one or more amino acids is within the weight percentage range of 15% - 25%.

32. The micro-particle composition of any one of claims 16-31, wherein the PVP is between about 31.25 % and about 38.89 % by weight of the final composition.

33. The micro-particle composition of any one of claims 16-32, wherein the PVP is between about 31% and about 39% by weight of the final composition.

34. A method of preparing a composition of spray freeze dried micro-particles, the method comprising:

(a) spraying a liquid formulation comprising:

(i) a PVP polymer having an average molecular weight of at least 1,300 kDa;

(ii) one or more sugars or sugar alcohols;

(iii) one or more amino acids; and

(iv) an active ingredient; to form droplets by atomization;

(b) freezing the droplets in a cryogenic fluid to form powdered micro-particles; and

(c) removing residual moisture from the powdered micro-particles by freeze drying in a vacuum chamber.

35. The method of claim 34, wherein the liquid formulation is the formulation of any one of claims 1-15.

36. The method of claim 34 or 35, wherein the spraying in (a) has a flow rate of about 5 ml/minute or less.

37. The method of claim 34, 35, or 36, wherein the freeze drying of (cj is for 4 hours at 300 mTorr and 20 hours at 100 Torr.

38. The method of any one of claims 34-37, wherein the freeze dried powdered micro- particles comprise an average diameter of about 100 μm to about 200 μm.

39. The method of any one of claims 34-38, wherein the freeze dried powdered micro- particles are suitable for intranasal administration.

40. Use of the micro-particle composition of any one of claims 16-33, for the treatment of a viral respiratory infection.

41. A method of treating a viral respiratory infection, comprising administering a micro- particle composition of any one of claims 16-33, to a subject in need thereof.

42. The micro-particle composition of any one of claims 16-33, for the treatment a viral respiratory infection.

43. Use of the micro-particle composition of any one of claims 16-33, for intranasal delivery of an active ingredient.

44. A method for intranasal delivery of an active ingredient, comprising administering a micro-particle composition of any one of claims 16-33 to a subject in need thereof.

45. The micro-particle composition of any one of claims 16-33, for intranasal delivery of an active ingredient.