Hyperbranched polyether polyols, methods for their preparation and uses thereof

Abstract

The present disclosure includes hyperbranched polyether polyols, such as hyperbranched poly(3-(oxiran-2-ylmethoxy) propane-1,2-diol) (HPOD), methods for their preparation and uses thereof, for example, as a cryoprotectant, lyoprotectant or stabilizer.

Description

HYPERBRANCHED POLYETHER POLYOLS, METHODS FOR THEIR PREPARATION AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority from co-pending U.S. provisional application no. 63 / 660,023 filed on June 14, 2024, the contents of which are incorporated herein by reference in their entirety.FIELD

[0002] The present disclosure relates, for example, to hyperbranched polyether polyols, such as hyperbranched poly(3-(oxiran-2-ylmethoxy) propane- 1,2-diol) (HPOD), methods for their preparation and uses thereof, for example, as a cryoprotectant, lyoprotectant or stabilizer.BACKGROUND

[0003] Biotherapeutics, including recombinant therapeutic proteins, hormones, monoclonal antibodies, enzymes, and growth factors represent a major portion of the current pharmaceutical market (Chennamsetty et al., 2009; Leader et al., 2008). Following the expedited and effective development of messenger RNA-based lipid nanoparticle (mRNA-LNP) vaccines in response to the emergence of SARS-CoV-2, there has been even greater momentum in the field of genetic medicines (Zhang et al., 2020; Cheng et al., 2022). Established as a vaccine against COVID-19, the mRNA-LNP technology is now being explored as a potential modality for future advancements in cancer treatment, genetic disorders, other infectious diseases, and CRISPR / Cas technologies which will likely increase the prevalence of LNPs in the field of gene medicine (Bevers et al., 2022; Eygeris et al., 2020; Zhang et al., 2022; Miller et al., 2017).

[0004] A major challenge to be tackled in this research field is the requirement for storage of active formulations at ultra-low temperatures (Schoenmaker et al., 2021). The LNPs as the crucial carrier system to preserve activity of nucleic acids, however, can burst, aggregate, or collapse during freeze-thaw cycles, meaning they require appropriate stabilization, preservation, and transportation protocols. Similarly, most protein-based biotherapeutic agents lose their activity upon exposure to external stimuli, such as temperature fluctuations, tight, desiccation, pH change, osmotic change, mechanical agitation, and hydrophobic surface interactions (Frokjaer & Otzen, 2005; Mitragotri et al., 2014). Currently, these pharmaceuticals are stored and preserved in formulations using excipients, such as amino acids, osmolytes, carbohydrates, and natural polymers (Arakawaet al., 2001). However, these excipients suffer from low efficiency, immunogenicity, high osmolarity, or interference with biological function (Kerwin, 2008; Shiraishi & Yokoyama, 2019). Since the inception of LNPs, enhancing their storage and stability has been a continuous priority, yet much about their structure remains to be elucidated. While significant strides have been made in understanding biotherapeutics and their stabilization (Panganiban et al., 2018; Liu et al., 2017; Marco-Dufort et al., 2022), many challenges remain. The challenges faced in the preservation of biotherapeutics and LNPs include toxicity (e.g., DMSO, glycerol) and difficulty in removing very high concentrations of cryoprotectants. Recently, there has been exploration into using macromolecule-based excipients, such as polymers, hydrogels, and zeolites, for storing protein-based biotherapeutics (Panganiban et al., 2018; Marco-Dufort et al., 2022; Liu et al., 2017; Herbert et al., 2021; Mancini et al., 2012). However, their biocompatibility and safety profiles remain undefined. While some carbohydrate-based systems have been investigated for preserving lipid nanoparticle (LNP)-based vaccines, macromolecular excipients have received less attention. Additionally, there is a lack of clinically viable lyophilization buffers for safely storing and transporting LNP-based therapeutics and vaccines. Thus, new (cryo)preserving molecules which are highly biocompatible and non-immunogenic are needed, while still delivering optimal performance to advance this important research field.

[0005] The use of lipid nanoparticle (LNP) carrier systems is useful for delivering charged nucleic acid cargo such as small interfering RNA (siRNA) (Kim et al., 2023), messenger RNA (mRNA) (Muramatsu et al., 2022) or self-amplifying RNA (saRNA) (Ball et al., 2017) into cells, where it can release its cargo to regulate gene expression or enable translation. While significant efforts have been focused on enhancing efficacy and stability of these formulations in vivo, there is still much to be explored and optimized in terms of storage conditions and particle preservation during storage (Haag et al., 2000). To ensure that the LNP remains intact during storage at ultralow temperatures, which are necessary for preserving RNA functionality, excipients are added to the formulation to retain the physicochemical properties of the delivery vehicle (Hassett et al., 2021; Di et al., 2022). While various studies have focused on enhancing LNP stability and efficacy through lipid component and formulation parameter optimization, failure to maintain these improvements during storage, distribution, and reconstitution may render the formulation noncompliant with necessary standards (Budke et al., 2014). The approved mRNA-based vaccine formulations from Comimaty™ (BioNTech / Pfizer) and Spikevax™ (Modema) use sucrose for preservation at 20% w / v in PBS and 8% w / v in Tris, respectively, which is considered safe.Comimaty is reported stable when stored at -70 °C for up to 6 months, and Spikevax is stored at -20 for up to 6 months (Deller et al., 2014; Kim et al., 2023). Sucrose is a highly osmotic compound which can cause damage to cells and tissue and thus, limits its use at high concentrations. Low concentrations, however, can lead to inadequate preservation, necessitating the need for alternatives possessing improved biocompatibility and lower osmolarity.

[0006] Lyophilization is an excellent preservation method compared to freezing, particularly for products that require long-term stability and preservation of their original structure and properties (Mancini et al., 2012). It can extend the shelf life of products and reduce storage and shipping costs because lyophilized products are lighter and take up less space than frozen products. The removal of water minimizes degradation of the product during storage and handling as water is the key factor for product degradation of biologically active samples; it can facilitate chemical reactions such as pH changes, osmotic changes, hydrolysis or oxidation, and promote enzymatic degradation.

[0007] The formation of a hydrogen-bond network amongst water molecules is directly associated with the crystallization during freezing. Therefore, molecules or polymers that can disrupt the hydrogen bonds between water molecules may have the potential to prevent the growth and formation of ice (Lee et al., 2022). Molecules possessing cavities or pockets that can accommodate water molecules exhibit distinct macroscopic properties, such as slowing ice crystal growth, non-colligative depression of freezing point, and inimitable dynamic structural, chemical, and surface properties (Biggs et al., 2017; Coyle et al., 1996; Hodge et al., 1996). Owing to their specific interactions with water, hydrated materials have been widely explored as stabilizing agents for proteins, enzymes (Keefe & Jiang, 2012), food ingredients (Griffith & Ewart, 1995), growth factors (Nguyen et al., 2013), skin hydrating products (Papakonstantinou et al., 2012), andfor cryopreservation of LNPs (Oude Blenke et al., 2023). Such studies are exemplified by excipients that are Generally Recognized as Safe (GRAS) by the FDA, such as disaccharides trehalose and sucrose (Ohtake & Wang, 2011) or polymers such as polyvinyl alcohol (Mitchell et al., 2019) and hyaluronic acid (Papakonstantinou et al., 2012). Despite the progress made in this field, the understanding of the design principles behind highly hydrating macromolecules remains limited (Biggs et al., 2017; Zheng et al., 2015).SUMMARY

[0008] The present disclosure includes a a hyperbranched poly ether polyol, comprising a combination of units selected from:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

[0009] In an embodiment, the hyperbranched polyether polyol comprises the:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

[0010] In an embodiment, the hyperbranched polyether polyol further comprises a core of the formula:5 whereinX1, X2and X3are each independently Ci-4alkylene; andR1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

[0011] In an embodiment, X1, X2and X3are all -CH2-.

[0012] In an embodiment, R1is ethyl.

[0013] In an embodiment, R1is the lipidic group. In another embodiment, the lipidic group is of the formula:wherein R1' is C4-3oalkyl.

[0014] The present disclosure also includes a method of preparing a hyperbranched polyether polyol, the method comprising polymerization of a compound of the structure:

[0015] In an embodiment, the method comprises anionic ring opening multi-branching polymerization (ROMBP). In another embodiment, the ROMBP comprises an initiator of the structure:whereinX1, X2and X3are each independently Ci-4alkylene; andR1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

[0016] In an embodiment, X1, X2and X3are all -CH2-.

[0017] In an embodiment, R1is ethyl.

[0018] In an embodiment, R1is the lipidic group. In another embodiment, the lipidic group is of the formula:, wherein R1' is C4-3oalkyl.

[0019] The present disclosure also includes a hyperbranched polyether polyol prepared by such methods of preparing a hyperbranched poly ether polyol.

[0020] In an embodiment, the hyperbranched poly ether polyol has a degree of branching of from about 1% to about 99%. In an embodiment, the hyperbranched poly ether polyol has a degree of branching of from about 23% to about 46%. The present disclosure also includes a hyperbranched polyether polyol having a degree of branching of from about 23% to about 46%. In an embodiment, the hyperbranched poly ether polyol has a degree of branching is about 23%, about 29%, about 37%, about 42% or about 46%. In an embodiment, the hyperbranched polyether polyol has a number average molecular weight (Mn) of from about 640 to about 11,500 Da. In an embodiment, the hyperbranched poly ether polyol has an intrinsic viscosity of from about 4 to about 15 mL / g. The present disclosure also includes a hyperbranched poly ether polyol having an intrinsic viscosity of from about 4 to about 15 mL / g.

[0021] The present disclosure also includes a conjugate comprising a hyperbranched polyether polyol of the present disclosure conjugated to a lipid.

[0022] In an embodiment, the lipid comprises a phospholipid.

[0023] The present disclosure also includes a lipid nanoparticle comprising (1) a hyperbranched polyether polyol of the present disclosure, wherein in the hyperbranched polyether polyol, R1is a lipidic group comprising at least 5 carbon atoms; or (2) a conjugate of the present disclosure. In an embodiment, the lipidic group is of the formula:, wherein R1' is C4-3oalkyl.

[0024] In an embodiment, the lipid nanoparticle further comprises an ionizable lipid, a phospholipid and cholesterol.

[0025] In an embodiment, the lipid nanoparticle further comprises an active ingredient.

[0026] The present disclosure also includes a liposome comprising (1) a hyperbranched polyether polyol of the present disclosure, wherein in the hyperbranched poly ether polyol, R1is a lipidic group comprising at least 5 carbon atoms; or (2) a conjugate of the present disclosure. In an embodiment, the lipidic group is of the formula:, wherein R1' is C4-3oalkyl.

[0027] In an embodiment, the liposome further comprises a phospholipid and cholesterol.

[0028] In an embodiment, the liposome further comprises an active ingredient.

[0029] The present disclosure also includes a composition comprising a hyperbranched polyether polyol or a conjugate of the present disclosure. In an embodiment, the composition further comprises an active ingredient. The present disclosure also includes a use of an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by such a composition. The present disclosure also includes a use of an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by such a composition. In an embodiment, the composition is a pharmaceutical composition, and the hyperbranched poly ether polyol or conjugate is not removed prior to use in the subject. In an embodiment, the use is of a pharmaceutical composition comprising the active ingredient encapsulated in or conjugated to a lipid nanoparticle comprising the hyperbranched poly ether polyol or conjugate of the present disclosure. In an embodiment, the subject is a human.

[0030] The present disclosure also includes a pharmaceutical composition comprising (1) an active ingredient and a hyperbranched poly ether polyol or a conjugate of the present disclosure; (2) a lipid nanoparticle of the present disclosure; or (3) a liposome of the present disclosure.

[0031] In an embodiment, the pharmaceutical composition comprises the hyperbranched polyether polyol of the present disclosure, the pharmaceutical composition further comprises a lipid nanoparticle and the active ingredient is encapsulated in or conjugated to the lipid nanoparticle. In an embodiment, the lipid nanoparticle comprises an ionizable lipid, a phospholipid, cholesterol and a linear PEG-lipid.

[0032] In an embodiment, the pharmaceutical composition comprises (1) the hyperbranched polyether polyol of the present disclosure, wherein in the hyperbranched polyether polyol, R1is a lipidic group comprising at least 5 carbon atoms; or (2) a conjugate of the present disclosure. In an embodiment, the lipidic group is of the formula:, wherein R1' is C4-3oalkyl.

[0033] The present disclosure also includes a use of a hyperbranched polyether polyol or conjugate of the present disclosure as a cryoprotectant, lyoprotectant or stabilizer or preservation agent or a hydrating agent or as an excipient. In an embodiment, the hyperbranched poly ether polyol or conjugate is for use in combination with an additional cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient. In an embodiment, the hyperbranched polyether polyol or conjugate is for use as the sole cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient. In an embodiment, the cryoprotection or lyoprotection is of an active ingredient. In an embodiment, the cryoprotection is of an organelle, a multicellular assembly, an organoid, a multicellular specimen, a cell, a tissue or an organ. In an embodiment, the stabilization is of a peptide or protein. In an embodiment, the peptide or protein is a biotherapeutic.

[0034] The present disclosure also includes a use of a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched polyether polyol and an active ingredient or a pharmaceutical composition comprising a hyperbranched poly ether polyol or conjugate of the present disclosure and an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject. The present disclosure also includes a use of a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched poly ether polyol and an active ingredient or a pharmaceutical composition comprising a hyperbranched polyether polyol or conjugate of the present disclosure and an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable by the active ingredient in a subject. In an embodiment, the lipid nanoparticle, liposome or pharmaceutical composition is cryopreserved then thawed prior to use in the subject. In an embodiment, the lipid nanoparticle, liposome or pharmaceutical composition is lyophilized and reconstituted prior to use in the subject. In an embodiment, the subject is a human.

[0035] In an embodiment, the active ingredient comprises a drug. In an embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In anembodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the ribonucleic acid comprises transfer RNA (tRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a protein or peptide.

[0036] The present disclosure also includes a use of a hyperbranched poly ether polyol or conjugate of the present disclosure as a food additive.

[0037] The present disclosure also includes a use of a hyperbranched poly ether polyol or conjugate of the present disclosure as a cosmetic additive.

[0038] The present disclosure also includes a food comprising a hyperbranched poly ether polyol or conjugate of the present disclosure as a food additive.

[0039] The present disclosure also includes a cosmetic comprising a hyperbranched polyether polyol or conjugate of the present disclosure as a cosmetic additive.

[0040] The present disclosure also includes the compound 3-(oxiran-2- ylmethoxy)propane-l,2-diol) (OPD). The present disclosure also includes the use of the compound OPD in the preparation of a hyperbranched polyether polyol.

[0041] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should rather be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The embodiments of the disclosure will now be described in greater detail with reference to the attached drawings, in which:

[0043] FIG. 1 shows a heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum (300 MHz, D2O) of 3-(oxiran-2-ylmethoxy) propane- 1,2-diol (OPD) monomer.

[0044] FIG. 2 shows a1H NMR spectrum (300 MHz, D2O) of hyperbranched poly 3- (oxiran-2-ylmethoxy)propane- 1,2-diol 3 (HPOD-3).

[0045] FIG. 3 shows an inverse gated13C NMR spectrum (75 MHz, D2O) of HPOD-3.

[0046] FIG. 4 shows a diffusion-ordered spectroscopy (DOSY) NMR spectrum (400 MHz, D2O) of HPOD-4 that was used to determine the diffusion coefficient of the HPOD.

[0047] FIG. 5 shows the gating strategy for cell transfection with messenger ribonucleic acid- lipid nanoparticle (mRNA-LNP) encoding for enhanced green fluorescent protein (EGFP).

[0048] FIG. 6 shows fluorescence intensity values of an insulin fibril formation assay determined by Thioflavin T for, from left to right: insulin (unheated), insulin (no preservative), and insulin preserved with trehalose, HPOD-2, HPOD-3, HPOD-4, polyethylene glycol (PEG), hyperbranched polyglycerol (HPG) and proline.

[0049] FIG. 7 shows synthesis of hyperbranched poly 3-(oxiran-2-ylmethoxy)propane- 1,2-diol (HPOD); a scheme of a representative synthetic route for HPOD by anionic ring opening multibranching polymerization (ROMBP) of 3-(oxiran-2-ylmethoxy)propane-l,2- diol (OPD). The possible different structural linkages within HPOD are shown (inserts).

[0050] FIG. 8 shows refractive index unit (RIU) traces of HPODs (1-5) from gel permeation chromatography (GPC) analysis.

[0051] FIGs. 9-10 show structural analysis to determine the degree of branching of HPODs. FIG. 9 shows13C inverse gated (upper) and distortionless enhancement by polarization transfer (DEPT; lower) NMR spectral analysis to identify the -CH (up) and -CH2 carbons with assigned structural units. FIG. 10 shows matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) spectra of HPOD-2 having multiples of 148 Da as main peaks which is equivalent to the molecular weight of the OPD monomer and peaks at a difference of 18 m / z indicating water adducts.

[0052] FIG. 11 shows MALDI-ToF spectra of HPG having a Mwof 1 kDa (upper) and of HPOD-2 (lower), showcasing different structural patterns made up from different monomers (74.2 Da indicating glycidol monomer and 148.3 Da indicating OPD monomer), different counter ions, and having water adducts (18 m / z).

[0053] FIG. 12 shows a MALDI-ToF spectrum of HPOD-1 showing a similar pattern as HPOD-2 (FIG. 11, lower). See Table 6 for calculations of the peaks and their weight composition.

[0054] FIG. 13 shows a total ion chromatogram (TIC, first image), an electrospray ionization (ESI) mass spectrum (second image) and a zoomed in portion of the ESI spectrum(third image) of HPOD-1 that shows the different monomer pattern as well as the water adducts 463.2 [m+H+H20], 467.2 [m+Na], and 485.2 [m+Na+ffcO].

[0055] FIG. 14 shows differential scanning calorimetry (DSC) thermograms of HPOD solutions (1-5) and pure water (upper) and polymers with similar molecular weight [HPOD, HPG, and polyglycerol dendrimer (PGD)] and trehalose (lower). HPOD, HPG, PGD, and trehalose were bound with 247, 209, 140, and 44.3 water molecules (hydration / molecule) respectively.

[0056] FIGs. 15-18 show dynamic light scattering (DLS) measurements of size in nm (FIG. 15 and FIG. 17) and poly dispersity index (PDI; FIG. 16 and FIG. 18) after cryostorage of mRNA-LNP under the indicated conditions overnight after one freeze-thaw cycle. Size and PDI were determined by DLS after freezing and compared to freshly prepared mRNA- LNPs [Fridge_Tris (FIGs. 15 and 16) / fridge_ 10% (FIGs. 17 and 18)].

[0057] FIGs. 19-21 show results from cryopreservation of RNA-LNP using HPODs. Freshly prepared mRNA-LNP were mixed with cryopreservation agents (HPOD, sucrose, trehalose, or buffer only (Tris buffer, 20 mM, pH 7)) and stored at -70 °C overnight. After thawing, HPODs showed significant improvement compared to unpreserved control in conservation of size (FIG. 19), PDI (FIG. 20) and encapsulation efficiency (EE%; FIG. 21). Data was reported for at least three independent experiments, *p<0.1, **p<0.01, ***p<0.001, vs original size, PDI and EE%, respectively).

[0058] FIG. 22 shows size (left axis) and PDI (right axis) for mRNA-LNP mixed with excipients and only stored at 4 °C overnight, without undergoing freeze-thaw cycles, for the indicated conditions. Both size and PDI were slightly increased for both HPOD-2 and HPOD- 5 compared to the original particles in buffer (Tris), while not wishing to be limited by theory, most likely due to association of the polymer with the lipid shell.

[0059] FIG. 23 shows results from cry opreservation of RNA-LNP using HPODs. Freshly prepared mRNA-LNP were mixed with the indicated cryopreservation agents or buffer only (Tris buffer, 20 mM, pH 7) and stored at -70 2 overnight. After thawing, HPODs showed significant improvement compared to unpreserved control (Tris buffer) in conservation of transfection in HeLa cells. Data was reported for at least three independent experiments, ****p<0.0001, vs original size %transfected cells.

[0060] FIG. 24 shows results from cry opreservation of RNA-LNP using HPOD-2; both mRNA (lower three columns) and self-amplifying (saRNA) integrity (upper three columns)within the LNPs, was measured after one freeze-thaw cycle on a bioanalyzer. HPOD-2 was compared to sucrose and unpreserved control (Tris buffer). %lost is the loss in integrity when compared to the integrity of freshly prepared fridge samples.

[0061] FIG. 25 shows results of when CHO cells were treated with formulations that had been prepared by mixing freshly prepared mRNA-LNPs encoding for enhanced green fluorescent protein (EGFP) with cryopreservation agents (HPOD-5 or trehalose) or buffer only (Tris)) and stored at -70 °C (center three columns) or lyophilized overnight (righthand three columns) then thawing / reconstitution of the respective formulation, and recordal of the percentage of transfected cells in comparison to LNPs prepared no longer than one week ago (LNP_fresh; far left column) and formulations that were stored in the fridge, prior to freezing or lyophilization (fridge_p20 and fridge_treh 20 1; second and third columns from left, respectively). HPOD-5 preserved the cell transfectability compared to the unpreserved control in CHO cells. Data is reported for three independent experiments.

[0062] FIG. 26 shows results of cell transfection in HeLa cells of EGFP transfecting mRNA-LNPs mixed with excipients HPOD-5 and trehalose or buffer only (Tris) after 1 and 3 freeze-thaw cycles over a total span of a week.

[0063] FIG. 27 shows results from cry opreservation of RNA-LNP using HPODs; confocal microscopy images of HeLa cells transfected with mRNA-LNPs encoding for enhEGFP, which were either preserved by HPOD-5 (right images) or remained unpreserved (Tris, 20 mM, pH 7; left images) during cryostorage at -70 °C (lower images) in comparison to freshly prepared mRNA-LNP that were just mixed and stored in the fridge (upper images). Scale bar is 100 pm.

[0064] FIG. 28 shows a schematic of potential degradation processes during freezing and lyophilization of mRNA-LNP (upper) and results from cryopreservation of mRNA-LNP using HPODs; i.m. injection into mice after cryostorage having been preserved with HPODs (lower; upper right image) or sucrose (lower; lower right image) or unpreserved (lower; lower left image) in comparison to freshly prepared mRNA-LNP (lower; upper left image).

[0065] FIG. 29 shows results from cryopreservation of RNA-LNP using HPODs; quantification of luminescence recorded in Balb / c mice 2d after i.m. injection of mRNA- LNP encoding for fLuc (second column from left) in comparison to freshly prepared mRNA-LNP (fridge_Tris), unpreserved (Tris), and preserved with sucrose (far right column). Results are shown as mean of n = 6 injections into 3 mice (* p < 0.1).

[0066] FIG. 30 shows size (upper plot, left axis), polydispersity index (PDI; upper plot, right axis) and encapsulation efficiency (lower plot) measurements for, from left to right in each plot: freshly prepared saRNA-LNPs, saRNA-LNPs unpreserved during cryostorage (Tris), saRNA-LNPs cryopreserved with HPOD-5 and saRNA-LNPs cryopreserved with sucrose. Values are presented after one freeze-thaw cycle and compared to original size, PDI, and EE%.

[0067] FIG. 31 shows results from cry opreservation of RNA-LNP using HPODs; 7 d after saRNA-LNP injection after they went through a freeze-thaw cycle (-70 °C). Results are compared to freshly prepared samples (fridge_Tris), samples that were unpreserved during cryostorage (Tris) and samples that were cryopreserved with sucrose (far right column) and are shown as mean of n = 6 injections into 3 mice (** p < 0.01).

[0068] FIGs. 32-36 show results of preservation during lyophilization of mRNA-LNP using HPOD. Freshly prepared mRNA-LNP were mixed with preservation agents HPOD-5 and sucrose or just buffer (Tris, 20 mM, pH 7) and lyophilized overnight. After reconstitution in water, particle size (FIG. 32), PDI (FIG. 33) and encapsulation efficiency were determined (FIG. 34). All samples showed significant size increase (####p<0.0001 vs original size (FIG. 32); but PDI (FIG. 33) and EE% (FIG. 34) remained the same (ns) for HPOD-5 (center data in each plot), outperforming sucrose (*p<0. 1 vs fridge PDI and####p<0.0001 vs original EE%; right values in each plot). Results are shown in comparison to unpreserved samples (Tris; left values in each plot). FIG. 35 shows that transfection in HeLa cells showed full preservation of transfectability by both HPOD-5 (center column) and sucrose (right column). Results are shown in comparison to unpreserved samples (left column). Data was reported for at least three independent experiments. FIG. 36 shows quantification of luminescence recorded in Balb / c mice 1 d after i.m. injection of mRNA-LNP encoding for fLuc for the conditions indicated. Results are shown as mean of n=6 injections into 3 mice (***p<0.001, **p<0.01).

[0069] FIGs. 37-41 show results of stabilization of proteins against heat, lyophilization, and freezing. FIG. 37 shows the activity of lysozyme after heating (60 °C, 30 min) in the presence of different HPODs and trehalose at 50 mg / mL. Lysozyme refers to the protein activity after heating without any excipients. HPOD-2, 3, and 4 showed enhanced activity compared to trehalose (n = 3,####p<0.0001). FIG. 38 shows screening of concentration range for trehalose and HPOD-4 for protection against heat shock at 60 °C of lysozyme. HPOD-4 showed concentration dependent activity whereas trehalose did not show any improved activity with concentration. Lysozyme refers to the protein activity after heating without anyexcipients. FIG. 39 shows preservation ability of HPODs against insulin aggregation upon heating (60 °C, 48 h) in comparison to different additives as indicated (n = 3). Insulin unheated from FIG. 6 was taken as the baseline signal, subtracted and normalized to and then plotted as % protection. The activity of the original insulin is also demonstrated, specifically referring to the absence of fibril formation. HPODs showed high efficiency in protecting insulin and were superior to the most commonly studied macro and small molecular additives, including trehalose, proline, and PEG (HPOD-2 vs trehalose (####p<0.0001), vs proline (###p = 0.0003), and vs PEG (###p = 0.0005)). All HPODs showed enhanced activity compared to trehalose (trehalose vs HPOD-2 (****p<0.0001), vs HPOD-3 (***p = 0.0003), and vs HPOD-4 (***p = 0.0009). FIG. 40 shows protection of / / -galactosidase from lyophilization stress using HPODs (n = 12). HPOD-2 and trehalose protected / / -galactosidase even at very low concentration (0.4 mg / mL). HPOD-2 showed improved performance at low concentrations compared to trehalose, however, there was no significant difference found at higher concentrations (trehalose vs HPOD-2 (0.4 mg / mL,####p<0.0001), (2 mg / mL,####p<0.0001), and (4 mg / mL, ns). FIG. 41 shows preservation of function of anti-blood group A antibody after freezing for 3 freeze-thaw cycles, where each cycle is performed within 24 h. The antibody activity after each freeze-thaw cycle is shown and the dotted line represents the activity of fresh antibody that did not undergo freezing and the bars represent the activity of stored antibodies mixed with the presented excipients. Trehalose is a known excipient and used as comparison for our HPOD excipients (N = 6 from 3 blood donors). Both HPOD-2 and HPOD-4 preserved antibody function after three repeated freeze-thaw cycles (one cycle every 24 h), and it is more efficient than trehalose (trehalose vs HPOD-4 (##p = 0.0030 (Day 1),##p =0.00545 (Day-2), and n.s. (Day 3)); trehalose vs HPOD-2 (###p =0.0005 (Day 1),##p =0.0029 (Day-2), and n.s. (Day 3)). Fresh antibody aliquot that never went through freezethaw cycles was used as a control. All the data are reported as mean ± s.d.

[0070] FIG. 42 shows screening of different macromolecules as indicated (50 mg / mL) for the protection of lysozyme at 60 °C. HPOD-2 (50 mg / mL) exhibited superior activity compared to other macromolecules<0.0001). The data was repeated for N = 3 and reported with mean ± s.d.

[0071] FIGs. 43-47 show HPODs (1 mg / mL) exhibited high viability in human umbilical vein endothelial cells (HUVECs; FIG. 43) and showed no adverse effects on blood coagulation as demonstrated by no change in activated partial thromboplastin time (APTT;FIG. 44) and prothrombin time (PT; FIG. 45). Heparin control (Heparinized PPP) did not clot at the given experimental conditions. Further, HPODs (1 mg / mL) did not induce any significant platelet activation (FIG. 46) or red blood cell (RBC) lysis (FIG. 47).

[0072] FIGs. 48-53 show biocompatibility of high concentration HPOD-4. HPOD-4 (40, 10, and 1 mg / mL final assay concentration) showed no adverse effects on blood coagulation as demonstrated by no change in activated partial thromboplastin time (aPTT; FIG. 48) and prothrombin time (PT; FIG. 49). Heparin and EDTA controls did not clot at the given experimental conditions. Further, HPOD-4 exhibited high viability in human endothelial cells fibroblast cells (BJ cells; FIG. 50) and EA.hy926 cells (FIG. 51). Finally, HPOD-4 did not induce any significant RBC lysis (FIG. 52) or platelet activation (FIG. 53) at any concentration.

[0073] FIGs. 54-59 show tolerance of HPOD in mice. There was no significant increase in aspartate amino transferase (AST; FIG. 54), alanine aminotransferase (ALT; FIG. 55), and lactate dehydrogenase (LDH; FIG. 56) levels compared to phosphate buffered saline (PBS; 1 x, pH 7.4) control as determined from mice sera taken on day 1 and day 14 after i.v. injection of HPOD at concentrations between 50 and 500 mg / kg. That indicates that HPOD was well tolerated in mice at high doses. FIG. 57 shows a schematic representation of the injection model and the organs collected. BALB / c mice were administered the excipient at a dose of up to 500 mg / kg of HPOD. The mice were monitored daily and body weights were observed. After 1 and 14 days, serum and organs were collected from sacrificed mice and analyzed. FIG. 58 is a plot showing that there was no change in body weight observed for mice injected with the excipient compared to mice injected with saline. FIG. 59 shows representative images of stained (H&E) organ slices (from left to right in each row: lung, heart, kidney, liver and spleen) collected from mice injected (i.v.) with 500 mg / kg HPOD (upper row) and PBS (1 x, pH 7.4; lower row). No abnormalities were seen in the heart, lungs, liver and kidneys of mice. All the data were reported with as mean ± s.d. of n = 4 mice per group.

[0074] FIGs. 60-64 show comparison of hydration of HPODs to polyglycerol polymers. FIG. 60 shows the influence of molecular weight and FIG. 61 shows the influence of degree of branching (DB) on the hydration of HPODs. With increase in molecular weight and DB, hydration of the HPODs was significantly decreased and reaches saturation rapidly when the molecular weight is >4000 Da as well as when DB is about 40%. FIG. 62 shows a schematic representation of chemical structures of three macromolecules; HPOD (left), hyperbranched polyglycerol (HPG; center), and polyglycerol dendrimer (PGD; right) with varying degrees ofbranching 40, 57, and 100% respectively. The number of dendritic units (dashed circles) and the nature of liner units (solid squares) of HPOD are quite different from HPG. FIG. 63 shows cryomicroscopic images of ice crystals formed in the presence of polymers after annealing at - 9.1 °C. The samples [1 mM solutions ofHPOD-4 (HPO; bottom images), PVA (8 kDa; second from bottom), and HES (180 kDa; second from top) in 30% sucrose and sucrose without any additives (top images)] were cooled to -50 °C to form small ice crystals (about 15 pm radii) and then annealed at -9. 1 °C while recording images of the ice crystals during annealing (scale bar is 100 pm). A representative set of images recorded after annealing at -9. 1 °C for 0 (left images) and 120 (right images) min are shown here. FIG. 64 is a plot showing the size of crystals represented here as average radii of 45 crystals. This study shows that HPOD works in a similar matter to HES but differs from PVA in its influence on the ice recrystallization inhibition.

[0075] FIGs. 65-66 show radius (FIG. 65) and cryomicroscopic images (FIG. 66) of ice crystals formed in the presence of additives at different concentrations in 30% sucrose solution after annealing at -9.1 °C. A representative set of enlarged images recorded after annealing at -9. 1 °C for 120 min are shown in FIG. 66 (scale bar in each image is 100 pm).

[0076] FIG. 67 shows results of cry opreservation studies of RBCs using HPODs; packed RBCs (80% hematocrit) were mixed with cry opreservation agents (HPOD, HES, and glycerol (175 mg / mL)) and stored in liquid nitrogen vapor for one week. HPODs showed significant improvement compared to saline control. HPOD-4 had enhanced activity; however, it is not significantly different from the rest (data reported for three independent experiments, mean ±sd , ****p<0.0001, glycerol, HPOD-4, and HES vs. saline).

[0077] FIG. 68 shows additional cry opreservation data that shows HPODs cryoprotect RBCs (80% hematocrit) similar to commonly studied additives, such as PVA and FDA approved agents (HES and glycerol). HPOD-4 showed better performance than HPOD-2, and both showed superior performance over saline control (HPOD-2 and 4 vs saline (****p < 0.0001), HPOD-4 vs HPOD-2 (*p = 0.0458)). Data are reported for three independent experiments, mean ± s.d.

[0078] FIGs. 69-71 show characterization of LNPs, SM-102 / DOPE / Chol / DMG-PEG-2000 or C 16HPOD with saRNA and N / P = 10. FIG. 69 is a plot comparing size (nm). FIG. 70 is a plot comparing zeta potential (mV). FIG. 71 is a plot comparing encapsulation efficiency (%).

[0079] FIGs. 72-75 show characterization of LNPs, MC3 / DSPC / Chol / DMG-PEG-2000 or C16HPOD with saRNA and N / P = 10. FIG. 72 is a plot comparing size (nm). FIG. 73 is a plot comparing zeta potential (mV). FIG. 74 is a plot comparing encapsulation efficiency (%).

[0080] FIG. 76 shows successful detection of lipo-polymer (C 16HPOD) in LNP by HPLC. LNP Formulations: SM-102 / DOPE / Chol / C I6HPOD (45:17.5:36.25:1.25) with saRNA and N / P = 10.

[0081] FIGs. 77-78 show size of liposomes with DMG-PEG-2000 or C16HPOD. FIG. 77 shows a comparison for DSPC / Chol. FIG. 78 shows a comparison for DOPC / Chol.DETAILED DESCRIPTIONI, Definitions

[0082] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the disclosure herein described for which they would be understood to be suitable by a person skilled in the art.

[0083] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process / method steps. As used herein, the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’ and any form thereof, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0084] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0085] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0086] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0087] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the numerical prefix “Cni-n2” For example, the term Ci-4alkyl means an alkyl group having 1, 2, 3 or 4 carbon atoms.

[0088] The term “alkylene” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkylene group, that is, a saturated carbon chain that contains substituents on two of its ends. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the numerical prefix “Cni-n2” For example, the term Ci-4alkylene means an alkylene group having 1, 2, 3 or 4 carbon atoms.

[0089] The term “subject” as used herein includes all members of the animal kingdom including mammals. In an embodiment, the subject is a human.

[0090] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example, mammals such as humans.

[0091] The terms “to treat”, “treating” and “treatment” and the like as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to alleviation or amelioration of one or more symptoms of a disease, disorder or condition treatable by the active ingredient, stabilized (i.e., not worsening) of the disease, disorder or condition treatable by the active ingredient, delay or slowing of the progression of the disease, disorder or condition treatable by the active ingredient, amelioration or palliation of the disease state of the disease, disorder or condition treatable by the active ingredient, diminishment of the reoccurrence of the disease, disorder or condition treatable by the active ingredient, and / or remission (whether partial or total) of the disease, disorder or condition treatable by the active ingredient, whether detectable or undetectable. “To treat”, “treating” and “treatment” and the like as used herein also include prophylactic treatment of the disease, disorder or condition treatable by the active ingredient. For example, a subject with an early stage of the disease, disorder or condition treatable by the active ingredient is treated to prevent progression or alternatively a subject in remission is treated to prevent recurrence.

[0092] The active ingredient, is for example, administered to the subject or used in an “effective amount”. As used herein, the term “effective amount” and the like means an amount effective, at dosages and for periods of time necessary to achieve a desired result. Desired results may depend, for example, on the identity of the disease, disorder or condition but would be readily understood by a person skilled in the art. Effective amounts may vary according to factors such as the state of the disease, disorder or condition, age, sex, weight and / or species of the subject. The amount of a given active ingredient that will correspond to such an amount will vary depending upon various factors, such as the given active ingredient, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder being treated, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.II. Hyperbranched Poly ether Polyols, Methods for their Preparation and Uses Thereof

[0093] RNA and protein-based therapeutics constitute almost half of recent drug approvals and capture considerable attention within biotechnology industries. Ensuring their stability and longevity in the context of heat, freezing, and lyophilization processes are paramount to a successful deployment. However, the advancement of formulations designed to achieve this goal is still in its nascent phase. It is an object of the present disclosure to address these challenges, and reported herein is a new class of semi-dendritic hydrophilic polymers with extended linear units, which showcase very high hydration. These polymers demonstrated exceptional efficacy in preserving mRNA- and saRNA-lipid nanoparticles during freezing and lyophilization. Additionally, they were found to protect therapeutic proteins against external stressors such as freezing, heat, and lyophilization. These polymers were also found to act as a cryoprotectant for red blood cells. These polymers are non-toxic, which enables their utilization at high concentrations and eliminates the requirement for removal prior to administration. Their unique topology contributes to the high hydration. By using initiators comprising a lipophilic group, hyperbranched lipo-polymers were prepared, which may, for example, be useful as an alternative to a linear PEG lipid in lipid nanoparticles and / or liposomes. These excipients are anticipated to create new prospects in biotechnology, food science, and cryopreservation.

[0094] Accordingly, the present disclosure includes a hyperbranched polyether polyol, comprising a combination of units selected from:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

[0095] The present disclosure also includes a hyperbranched polyether polyol, comprising:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

[0096] A person skilled in the art would readily appreciate that such hyperbranched polyether polyols further comprise a core, the structure of which may depend, for example, on the nature of the method used to prepare the hyperbranched poly ether polyol. The core can be any suitable core. In an embodiment, the core is of the formula:5 whereinX1, X2and X3are each independently Ci-4alkylene; andR1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

[0097] In an embodiment, X1, X2and X3are each independently Ci-2alkylene. In another embodiment, X1, X2and X3are all -CH2-.

[0098] In an embodiment, R1is C2-3alkyl. In another embodiment, R1is ethyl.

[0099] In an embodiment, the core is of the formula:

[0100] In an embodiment, R1is the lipidic group. The term “lipidic group” as used herein includes lipidic groups that are at least substantially, optionally fully hydrophobic as well as amphiphilic lipidic groups that comprise one or more hydrophobic tails and a hydrophilic head group. The term “lipidic group” as used herein also includes lipidic groups derived from natural sources as well as synthetic and semi-synthetic sources. The lipidic group can be any suitable lipidic group, the selection of which can be made by the person skilled in the art. In an embodiment, the lipidic group is derived from a fatty acid, a wax, a monoglyceride, a diglyceride, a triglyceride, a phospholipid, a sphingolipid, an amino lipid or a sterol. In an embodiment, the lipidic group is attached to the remainder of the core via an alkyl ether linkage (e.g., a CH2-O- linkage). However, the skilled person would appreciate that any suitable linkage may be used. In an embodiment, the lipidic group is of the formula:wherein R1' is C4-3oalkyl.

[0101] In an embodiment, R1is Ci2-3oalkyl. In another embodiment, R1is Ci6-24alkyl. In an embodiment, R1is linear alkyl. In another embodiment, R1is branched alkyl.

[0102] In an embodiment, the lipidic group is of the formula:

[0103] In another embodiment, the lipidic group is of the formula:

[0104] The present disclosure also includes a method of preparing a hyperbranched polyether polyol, the method comprising polymerization of a compound of the structure:O OH^V / O^A^OH

[0105] The polymerization can comprise any suitable method, the selection of which can be made by a person skilled in the art. In an embodiment, the polymerization comprises anionic ring opening multi -branching polymerization (ROMBP). The conditions for the ROMBP can comprise any suitable conditions, the selection of which could be made by a person skilled in the art having regard to the present disclosure.

[0106] In an embodiment, the ROMBP comprises an initiator of the structure:whereinX1, X2and X3are each independently Ci-4alkylene; andR1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

[0107] In an embodiment, X1, X2and X3are each independently Ci-2alkylene. In another embodiment, X1, X2and X3are all -CH2-.

[0108] In an embodiment, R1is C2-3alkyl. In another embodiment, R1is ethyl.

[0109] In an embodiment, the initiator is of the formula:

[0110] In an embodiment, R1is the lipidic group. The lipidic group can be any suitable lipidic group, the selection of which can be made by the person skilled in the art. In an embodiment, the lipidic group is derived from a fatty acid, a wax, a monoglyceride, adiglyceride, a triglyceride, a phospholipid, a sphingolipid, an amino lipid or a sterol. In another embodiment, the lipidic group is attached to the remainder of the core via an alkyl ether linkage (e.g., a CH2-O- linkage). However, the skilled person would appreciate that any suitable linkage may be used. In an embodiment, the lipidic group is of the formula:wherein R1is C4-3oalkyl.

[0111] In an embodiment, R1is Ci2-3oalkyl. In another embodiment, R1is Ci6-24alkyl. In an embodiment, R1is linear alkyl. In another embodiment, R1is branched alkyl.

[0112] In an embodiment, the lipidic group is of the formula:

[0113] In another embodiment, the lipidic group is of the formula:

[0114] In an embodiment, the method comprises mixing the initiator with a suitable base (e.g., potassium methoxide) in methanol at a suitable temperature (e.g., room temperature (e.g., from about 4°C to about 40°C or about 21°C to about 25°C)) for a suitable time (e.g., about 15 minutes to about 1 hour or about 30 minutes), then evaporating a suitable amount of methanol (e.g., at a temperature of about 70°C for a time of about 2 hours to about 6 hours or about 4 hours), then raising the temperature (e.g., to about 90°C), adding the compound of the structure:and mixing for a suitable amount of time (e.g., about 2 hours to about 12 hours or about 5 hours) followed by quenching with a suitable acid (e.g., HC1) to obtain a neutral pH.

[0115] The ratio of the initiator : base : compound can be any suitable ratio, the selection of which can be made by a person skilled in the art having regard to the present disclosure. In an embodiment, the ratio of the initiator : base : compound is about 1 : 0.3 : 6, about 1 : 0.1 : 40, about 1 : 0.4 : 34, about 1 : 0.33 : 20 or about 1 : 0.4 : 80. In another embodiment, the ratio of the initiator : base : compound is about 1 : 0.3 : 6. In anotherembodiment, the ratio of the initiator : base : compound is about 1 : 0.1 : 40. In another embodiment, the ratio of the initiator : base : compound is about 1 : 0.4 : 34. In another embodiment, the ratio of the initiator : base : compound is about 1 : 0.33 : 20. In another embodiment, the ratio of the initiator : base : compound is about 1 : 0.4 : 80.

[0116] In an embodiment, the method further comprises purifying the crude hyperbranched polyether polyol. In an embodiment, the purification comprises precipitation of the crude hyperbranched polyether polyol. In an embodiment, the purification further comprises dialysis of the precipitated hyperbranched poly ether polyol. In an embodiment, the purification comprises dialysis of the crude hyperbranched poly ether polyol.

[0117] The present disclosure also includes a hyperbranched poly ether polyol prepared by a method of preparing a hyperbranched poly ether polyol of the present disclosure.

[0118] In an embodiment, the hyperbranched polyether polyols of the present disclosure have a degree of branching of from about 1% to about 99%. In an embodiment, the hyperbranched poly ether polyol of the present disclosure has a degree of branching of from about 23% to about 46%. Accordingly, the present disclosure also includes a hyperbranched poly ether polyol having a degree of branching of from about 23% to about 46%. In another embodiment, the degree of branching of the hyperbranched poly ether polyols of the present disclosure is about 23%, about 29%, about 37%, about 42% or about 46%. In another embodiment, the degree of branching of the hyperbranched poly ether polyols of the present disclosure is about 23%. In another embodiment, the degree of branching of the hyperbranched polyether polyols of the present disclosure is about 29%. In another embodiment, the degree of branching of the hyperbranched poly ether polyols of the present disclosure is about 37%. In another embodiment, the degree of branching of the hyperbranched polyether polyols of the present disclosure is about 42%. In another embodiment, the degree of branching of the hyperbranched poly ether polyols of the present disclosure is about 46%. In another embodiment, the degree of branching of the hyperbranched poly ether polyols of the present disclosure is from about 23% to about 29%.

[0119] In an embodiment, the hyperbranched polyether polyols of the present disclosure have a number average molecular weight (Mn) of from about 640 to about 11,500 Da. However, hyperbranched polyether polyols of the present disclosure of significantly higher molecular weights may be prepared. In another embodiment, the hyperbranched polyether polyols of the present disclosure have a number average molecular weight (Mn) of less than about 4,000.

[0120] In an embodiment, the hyperbranched polyether polyols of the present disclosure have an intrinsic viscosity of from about 4 to about 15 mL / g.

[0121] The present disclosure also includes a hyperbranched polyether polyol having an intrinsic viscosity of from about 4 to about 15 mL / g.

[0122] The present disclosure also includes a conjugate comprising a hyperbranched polyether polyol of the present disclosure conjugated to a lipid. The lipid can be any suitable lipid, the selection of which can be made by the person skilled in the art. In some embodiments, the lipid is at least substantially, optionally fully hydrophobic. In some embodiments, the lipid is an amphiphilic moiety that comprises one or more hydrophobic tails and a hydrophilic head group. The lipid may be derived from natural sources or synthetic and semi-synthetic sources. In an embodiment, the lipid is a fatty acid, a wax, a monoglyceride, a diglyceride, a triglyceride, a phospholipid, a sphingolipid, an amino lipid or a sterol. In another embodiment, the lipid is a phospholipid.

[0123] The present disclosure also includes a lipid nanoparticle comprising (1) a hyperbranched poly ether polyol of the present disclosure comprising a core of the formula:5 whereinX1, X2and X3are each independently Ci-4alkylene; andR1is a lipidic group comprising at least 5 carbon atoms; or a hyperbranched polyether polyol of the present disclosure prepared from a method comprising ROMBP comprising an initiator of the structure:whereinX1, X2and X3are each independently Ci-4alkylene; andR1is a lipidic group comprising at least 5 carbon atoms; or(2) a conjugate comprising a hyperbranched polyether polyol of the present disclosure conjugated to a lipid.

[0124] It will be readily appreciated by a person skilled in the art that in embodiments relating to such lipid nanoparticles, the hyperbranched polyether polyols may be varied as described herein in other embodiments except that R1is the lipidic group.

[0125] In an embodiment, the lipid nanoparticles comprise the hyperbranched polyether polyol. In another embodiment, the lipid nanoparticles comprise the conjugate.

[0126] The lipid nanoparticles can comprise any suitable components in addition to the hyperbranched polyether polyol or conjugate of the present disclosure. In an embodiment, the lipid nanoparticle further comprises an ionizable lipid, a phospholipid and cholesterol. The ionizable lipid can be any suitable ionizable lipid, the selection of which can be readily made by a person skilled in the art. The phospholipid can be any suitable phospholipid, the selection of which can be readily made by a person skilled in in the art. In an embodiment, the ionizable lipid comprises an amino lipid (e.g., SM-102 or MC3). In another embodiment, the phospholipid comprises a phosphoethanolamine (e.g., 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE)) or a phosphatidylcholine (e.g., distearoylphosphatidylcholine (DSPC)). In another embodiment, the phospholipid comprises a phosphoethanolamine. In another embodiment, the phospholipid comprises a phosphatidylcholine. In an embodiment, the lipid nanoparticle comprises SM-102 and DOPE. In another embodiment, the lipid nanoparticle comprises MC3 and DSPC.

[0127] In an embodiment, the lipid nanoparticle further comprises an active ingredient. In an embodiment, the active ingredient comprises a drug. In another embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the active ingredient comprises messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a peptide or protein. In an embodiment, the peptide or protein is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor. In an embodiment, the active ingredient is encapsulated within the lipid nanoparticle. In another embodiment, the active ingredient is conjugated to the lipid nanoparticle.

[0128] The present disclosure also includes a liposome comprising (1) a hyperbranched polyether polyol of the present disclosure comprising a core of the formula:5 whereinX1, X2and X3are each independently Ci-4alkylene; andR1is a lipidic group comprising at least 5 carbon atoms; or a hyperbranched polyether polyol of the present disclosure prepared from a method comprising ROMBP comprising an initiator of the structure:whereinX1, X2and X3are each independently Ci-4alkylene; andR1is a lipidic group comprising at least 5 carbon atoms; or(2) a conjugate comprising a hyperbranched polyether polyol of the present disclosure conjugated to a lipid.

[0129] It will be readily appreciated by a person skilled in the art that in embodiments relating to such liposomes, the hyperbranched polyether polyols may be varied as described herein in other embodiments except that R1is the lipidic group.

[0130] In an embodiment, the liposome comprises the hyperbranched poly ether polyol. In another embodiment, the liposome comprises the conjugate.

[0131] The liposomes can comprise any suitable components in addition to the hyperbranched polyether polyol of the present disclosure. In an embodiment, the liposome further comprises a phospholipid and cholesterol. The phospholipid can be any suitable phospholipid, the selection of which can be readily made by a person skilled in in the art. In an embodiment, the phospholipid comprises a phosphatidylcholine (e.g., 1 ,2-dioleoyl-sn-glycero- 3-phosphocholine (DOPC) or distearoylphosphatidylcholine (DSPC)). In another embodiment, the phospholipid comprises DOPC. In another embodiment, the phospholipid comprises DSPC.

[0132] In an embodiment, the liposome further comprises an active ingredient. In an embodiment, the active ingredient comprises a drug. In another embodiment, the activeingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the active ingredient comprises messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a peptide or protein. In an embodiment, the peptide or protein is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor. In an embodiment, the active ingredient is encapsulated within the liposome. In another embodiment, the active ingredient is conjugated to the liposome.

[0133] The present disclosure also includes a composition comprising a hyperbranched polyether polyol or conjugate of the present disclosure. In an embodiment, the composition comprises the hyperbranched polyether polyol. In an embodiment, the composition comprises the conjugate. In an embodiment, the composition further comprises an active ingredient. In an embodiment, the active ingredient comprises a drug. In another embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the active ingredient comprises messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a protein or peptide. In an embodiment, the protein or peptide is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor. The present disclosure also includes a use of an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by such a composition. The present disclosure also includes a use of an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable bythe active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by such a composition. The present disclosure also includes an active ingredient for use in the treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by such a composition. The present disclosure also includes a method for treating a disease, disorder or condition treatable by an active ingredient in a subject, the method comprising cryoprotecting, lyoprotecting or stabilizing the active ingredient by such a composition, and administering the active ingredient to the subject. In an embodiment, the composition is a pharmaceutical composition, and the hyperbranched poly ether polyol or conjugate, as the case may be, is not removed prior to use in / admini strati on to the subject.

[0134] The present disclosure also includes a pharmaceutical composition comprising an active ingredient and (1) a hyperbranched poly ether polyol of the present disclosure or a conjugate of the present disclosure; (2) a lipid nanoparticle of the present disclosure; or (3) a liposome of the present disclosure. In an embodiment, the active ingredient comprises a drug. In another embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (rnRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the ribonucleic acid comprises messenger RNA (mRNA), selfamplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a protein or peptide. In an embodiment, the protein or peptide is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor. In an embodiment, the pharmaceutical composition comprises the hyperbranched polyether polyol of the present disclosure. In an embodiment, the pharmaceutical composition comprises the hyperbranched polyether polyol of the present disclosure, the pharmaceutical composition further comprises a lipid nanoparticle and the active ingredient is encapsulated in or conjugated to the lipid nanoparticle. In an embodiment, the active ingredient is encapsulated within the lipid nanoparticle. In another embodiment, the active ingredient is conjugated to the lipid nanoparticle. The lipid nanoparticle can comprise any suitable lipid nanoparticle. In anembodiment, the lipid nanoparticle comprises an ionizable lipid, a phospholipid, cholesterol and a linear PEG lipid. In another embodiment, the pharmaceutical composition comprises the conjugate of the present disclosure. In an embodiment, the pharmaceutical composition comprises the lipid nanoparticle of the present disclosure. In an embodiment, the active ingredient is encapsulated within the lipid nanoparticle of the present disclosure. In another embodiment, the active ingredient is conjugated to the lipid nanoparticle of the present disclosure. In another embodiment, the pharmaceutical composition comprises the liposome of the present disclosure. In an embodiment, the active ingredient is encapsulated within the liposome of the present disclosure. In another embodiment, the active ingredient is conjugated to the liposome of the present disclosure

[0135] The present disclosure also includes a use of a hyperbranched polyether polyol of the present disclosure or conjugate of the present disclosure as a cryoprotectant, lyoprotectant or stabilizer or preservative agent or hydrating agent or excipient. In an embodiment, the use is as a cryoprotectant, lyoprotectant or stabilizer. In an embodiment, the use is as a cryoprotectant. In another embodiment, the use is as a lyoprotectant. In an embodiment, the use is as a stabilizer. In an embodiment, the use is as a preservative agent. In an embodiment, the use is as a hydrating agent. In an embodiment, the use is as an excipient. In an embodiment, the hyperbranched poly ether polyol or conjugate is for use in combination with an additional cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient. In another embodiment, the hyperbranched poly ether polyol or conjugate is for use as the sole cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient. In an embodiment, the use is of the hyperbranched poly ether polyol. In another embodiment, the use is of the conjugate.

[0136] In an embodiment, the cryoprotection or lyoprotection is of an active ingredient. In an embodiment, the active ingredient comprises a drug. In another embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the ribonucleic acid comprises messenger RNA (mRNA), self-amplifyingRNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a protein or peptide. In an embodiment, the protein or peptide is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor.

[0137] In an embodiment, the cryoprotection is of a cell, multicellular assembly, an organoid, a multicellular specimen, a tissue or an organ. In an embodiment, the cryoprotection is of a cell. In another embodiment, the cell is a red blood cell.

[0138] In an embodiment, the stabilization is of a peptide or protein. In an embodiment, the peptide or protein is a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor. In an embodiment, the stabilization is against heat.

[0139] The present disclosure also includes a use of a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched polyether polyol and an active ingredient or a pharmaceutical composition comprising a hyperbranched poly ether polyol or conjugate of the present disclosure and an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject. The present disclosure also includes a use of a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched poly ether polyol and an active ingredient or a pharmaceutical composition comprising a hyperbranched polyether polyol or conjugate of the present disclosure and an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable by the active ingredient in a subject. In an embodiment, the use is of the lipid nanoparticle of the present disclosure and the active ingredient. In another embodiment, the use is of the liposome of the present disclosure and the active ingredient. In another embodiment, the use is of the pharmaceutical composition comprising the hyperbranched polyether polyol of the present disclosure. In another embodiment, the use is of the pharmaceutical composition comprising the conjugate of the present disclosure. The present disclosure also includes a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched polyether polyol and an active ingredient or a pharmaceutical composition comprising a hyperbranched polyether polyol or conjugate of the present disclosure and an active ingredient for use in the treatment of a disease, disorder or condition treatable by the active ingredient in a subject. In an embodiment, the lipid nanoparticle of the present disclosure and the active ingredient are for the use. In an embodiment, the liposome of the present disclosure and the active ingredient arefor the use. In another embodiment, the pharmaceutical composition comprising the hyperbranched poly ether polyol of the present disclosure is for the use. In another embodiment, the pharmaceutical composition comprising the conjugate of the present disclosure is for the use. The present disclosure also includes a method for treating a disease, disorder or condition treatable by an active ingredient in a subject, the method comprising administering a lipid nanoparticle or liposome of the present disclosure comprising a hyperbranched poly ether polyol and the active ingredient or a pharmaceutical composition comprising a hyperbranched poly ether polyol or conjugate and the active ingredient to the subject. In an embodiment, the method comprises administration of the lipid nanoparticle of the present disclosure and the active ingredient. In another embodiment, the method comprises administration of the liposome of the present disclosure and the active ingredient. In another embodiment, the method comprises administration of the pharmaceutical composition comprising the hyperbranched polyether polyol of the present disclosure. In another embodiment, the method comprises administration of the pharmaceutical composition comprising the conjugate of the present disclosure. It will be appreciated by a person skilled in the art that such lipid nanoparticles and liposomes of the present disclosure may be formulated into pharmaceutical compositions for administration to subjects or use in a biologically compatible form suitable for administration or use in vivo.

[0140] In an embodiment, the lipid nanoparticle, liposome or pharmaceutical composition of the present disclosure is cryopreserved then thawed prior to use in / administration to the subject. In an embodiment, the lipid nanoparticle, liposome or pharmaceutical composition of the present disclosure is lyophilized and reconstituted prior to use in the subject.

[0141] In an embodiment, the active ingredient comprises a drug. In another embodiment, the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO). In an embodiment, the active ingredient comprises the nucleic acid. In an embodiment, the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In an embodiment, the active ingredient comprises a ribonucleic acid (RNA). In another embodiment, the active ingredient comprises transfer (RNA), messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In another embodiment, the ribonucleic acid comprises messenger RNA (mRNA), self-amplifying RNA (saRNA) or small interfering RNA (siRNA). In an embodiment, the active ingredient comprises a protein or peptide. In an embodiment, the protein or peptideis a biotherapeutic. In an embodiment, the biotherapeutic comprises a recombinant therapeutic protein, hormone, monoclonal antibody, enzyme or growth factor.

[0142] In an embodiment, the pharmaceutical composition comprises a lipid nanoparticle of the present disclosure comprising a hyperbranched polyether polyol and the active ingredient is encapsulated in or conjugated to the lipid nanoparticle. In an embodiment, the active ingredient is encapsulated within the lipid nanoparticle. In another embodiment, the active ingredient is conjugated to the lipid nanoparticle. In an embodiment, the pharmaceutical composition comprises a liposome of the present disclosure comprising a hyperbranched poly ether polyol and the active ingredient is encapsulated in or conjugated to the liposome. In an embodiment, the active ingredient is encapsulated within the liposome. In another embodiment, the active ingredient is conjugated to the liposome.

[0143] A person skilled in the art would readily appreciate the diseases, disorders or conditions treatable by a particular active agent.

[0144] Treatment methods or uses comprise administering to a subject or use of an effective amount of an active agent as described herein, optionally consisting of a single administration or use, or alternatively comprising a series of administrations or uses. For example, the active agent is administered or used at least once a week. However, in another embodiment, the active agent is administered to the subject or used from one time per three weeks, or one time per week to once daily for a given treatment or use. In another embodiment, the active agent is administered or used 2, 3, 4, 5 or 6 times daily. The length of the treatment period or use depends on a variety of factors, such as the identity of the disease, disorder or condition treatable by the active agent, the severity of the disease, disorder or condition treatable by the active agent, the age of the subject, the concentration of the active agent in a / the pharmaceutical composition, the activity of the active agent and / or a combination thereof. It will also be appreciated that the effective amount of the active agent may increase or decrease over the course of a particular treatment regime or use. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration or use is required. For example, the active agent is administered or used in an amount and for duration sufficient to treat the subject.

[0145] The present disclosure also includes a use of a hyperbranched poly ether polyol or conjugate of the present disclosure as a food additive. The present disclosure also includes a use of a hyperbranched polyether polyol or conjugate of the present disclosure as a cosmeticadditive. In an embodiment, the use is of the hyperbranched polyether polyol. In an embodiment, the use is of the conjugate. The present disclosure also includes a food comprising a hyperbranched polyether polyol or conjugate of the present disclosure as a food additive. In an embodiment, the food comprises the hyperbranched poly ether polyol. In another embodiment, the food comprises the conjugate. The present disclosure also includes a cosmetic comprising a hyperbranched poly ether polyol or conjugate of the present disclosure as a cosmetic additive. In an embodiment, the cosmetic comprises the hyperbranched polyether polyol. In another embodiment, the cosmetic comprises the conjugate.

[0146] The present disclosure also includes the compound 3-(oxiran-2- ylmethoxy)propane-l,2-diol) (OPD). The present disclosure also includes the use of the compound OPD in the preparation of a hyperbranched polyether polyol. In an embodiment, the use is in a method of preparing a hyperbranched polyether polyol of the present disclosure.

[0147] The following are non-limiting examples of the present disclosure:EXAMPLESExample 1. An Ultrahydrating Polymer that Protects Protein Therapeutics and RNA- Lipid Nanoparticles Against Freezing, Heat and Lyophilization Stress.

[0148] Herein, the development of a class of semi-dendritic ultra-hydrating polymers, hyperbranched poly(3-(oxiran-2-ylmethoxy) propane- 1,2-diols (HPODs), that are highly effective in stabilizing diverse biotherapeutics, including mRNA-LNP, saRNA-LNP, proteins, enzymes, and antibodies against external stresses such as freezing, heating, and lyophilization compared to the current gold standards used in academia as well as in industry is reported. These branched polymers were well tolerated in vivo at high concentration, eliminating the need to be removed prior to administration. Moreover, the significance of polymer topology in determining their exceptional hydration properties was demonstrated, thereby establishing an additional design criterion alongside chemical composition.I. Experimental(a) General Methods

[0149] All reagents, solvents, and molecular sieves (4 A) were purchased from Sigma- Aldrich™, Canada, unless otherwise mentioned. Deuterated solvents (D2O and MeOD 99.8% D) were purchased from Cambridge Isotope Laboratories, Inc. Standard regenerated cellulose(RC) membranes (MWCO-O.5, 1, 3.5, and 5 kDa) for dialysis were purchased from Spectrum, Inc., USA. NMR spectra (XH,13C,13C inverse-gated (IG), HSQC, DEPT-135, and diffusion coefficient measurements (D)) of polymers were recorded on Bruker Avance™ 300 and 400 MHz spectrometers. The degree of branching (DB) of the polymers was calculated using the equation, DB = 2D+sD / (2D+(4 / 3)sD+2 / 3L), where D, sD and L represent the intensities of the signals corresponding to the dendritic, semidendritic and linear units respectively, which were determined using13C IG NMR spectroscopy (D2O, relaxation delay of 6 s) (Sunder et al., 1999; Imran ul-haq et al., 2013; Holter et al., 1997). The determination of the DB was based on approximation and theoretical probability of formation of structural units in AB3. Since identifying the actual structural linkages in HPOD using AB3 kinetics is a significant project on its own, the peaks were initially labelled using AB2 calculations (L13, D, 2L14, 2D, 2T, L13, L14, T, L13). These labeled peaks were then utilized to assess the probability of peaks forming according to AB3 calculations as shown in Scheme 1.Scheme 1. Structural Features Representation of different possible structural units of HPOD (A), HPG (B), and PGD (C).

[0150] These units were further categorized as D+T, L13+14, 2L14, D+L13, D+L14, 2D, L14+T, linkages. By considering the similar chemical nature of these linkages with AB2 structural linkages and the theoretical probability of formation of structural units in AB3 (T:L:SD:D at a probability of 8: 12:6: 1), the probability of formation for the remaining linkages (D, T, L13, and L14 linkages) was determined. Absolute molecular weight of the HPODs was determined by a Waters1Msize exclusion chromatography system fitted with a multi angle laser light scattering (MALS) detector (DAWN HELEOS II) and refractive index detector (Optilab T-rEX). Viscometer (Viscostar II) detector was used to measure the intrinsic viscosity of the polymers in a 0.1 N NaNCf buffer (pH = 7.0) mobile phase. All the detectors were from WyattTechnologies, Inc., Santa Barbara, CA. Gel permeation chromatography analysis was performed using Waters ultrahydrogel columns (guard, linear and 120) and 0. 1 NNaNCh buffer (pH = 7.0) was used as a mobile phase, and dn / dc value for HPODs was determined as 0.12 mL / g. Fusion enthalpy of polymer solutions and water was determined using differential scanning calorimetry (DSC) (Q2100, TA instruments, New Castle, DE, USA). All the DSC data was processed using TA instruments universal 2100 software. Thermite pans and lids were purchased from Perkin Elmer. Absorbance measurements were performed on SpectraMax™ M3 (Molecular Devices). Fluorescence measurements were carried out on a Cary Eclipse fluorescence spectrophotometer (Agilent™). Confocal imaging was carried out on a Nikon C2+Confocal Microscope System. Polyglycerol dendrimer (PGD) (generation 3) was synthesized and characterized by following a previous report (Haag et al., 2000).Synthesis of 3-(oxiran-2-ylmethoxy) propane- 1,2-diol (OPD) monomer

[0151] m-Chloroperbenzoic acid (m-CPBA; 37.6 grams, 0.168 mol, 1.5 eq, 77 wt%) was added in small portions to a solution of 3-allyloxy-l,2 propanediol (15 G, 0.112 mol) in dichloromethane (DCM; 250 mL) at room temperature (RT) and the reaction mixture was stirred for 20 h. A white precipitate, m-chlorobenzoic acid as a by-product, was noticed over time in the flask and the reaction mixture was filtered to remove the m-chlorobenzoic acid. The volume of the filtrate was slightly concentrated (about 25-30% of the original volume or until a small amount of precipitate was noticed in the flask) and the flask was kept at 0 °C to further precipitate and filter out the m-chlorobenzoic acid. The concentration-precipitation-filtration cycles were repeated three more times and a final 100-120 mL of DCM solution was washed with 100 mL of water 3 times. The water fraction also contained a small amount of m- chlorobenzoic acid, which was confirmed by1H NMR. To remove it, the collected 300 mL of aqueous fraction was washed three times with 100 mL of ethyl acetate (EtOAc). The obtained water layer was lyophilized until the water content (analyzed by thermogravimetiy analysis) in the monomer was about 1% (yield: 60-70%). The structure of the monomer was determined by NMR spectroscopy [H1NMR, C13NMR and HSQC (FIG. 1 )] and mass spectroscopy.1H NMR (400 MHz, D2O-d ): 52.78 (m, 1H), 2.96 (t, J = 4.44 Hz, 1H), 3.37 (m, 2H), 3.60 (m, 4H), 3.91 (m, 2H).13C NMR (75 MHz, D2O-d2) 45.01, 51.75, 62.64, 70.50, 72.07, 72.15. HRMS(TOF ES+): Calcd for C6Hi2O4: 148.07 [m / z], 171.07 [m+Na],Synthesis of hyperbranched poly (3-(oxiran-2-ylmethoxy) propane- 1,2-diol (HPOD) polymers

[0152] HPOD polymers were synthesized using anionic ring opening multibranching polymerization (Sunder et al., 1999; Imran ul-haq et al., 2013). A representative synthetic procedure for HPOD-3 is described as follows. Briefly, trimethylolpropane (82 mg, 0.61 mmol) was taken in a flame dried three neck flask and potassium methoxide (KOMe; 74 pL, in 25% MeOH) was added under argon, and stirred for 30 min at RT. After methanol was evaporated at 70 °C for 4 h, the reaction temperature was raised to 90 °C. The flask was connected to an overhead stirrer and OPD monomer (3.1 g, 21 mmol) was added slowly (0.25 mL / h) under argon using a syringe pump After addition was completed, the reaction mixture was stirred for an additional 5 h. The reaction mixture was quenched with a very small amount of 0.01 M HC1 to reach a neutral pH. The polymer was precipitated twice by dissolving in MeOH and precipitating with an excess amount of methanol (MeOH) / di ethyl ether (1:9 v / v). The precipitated polymer was purified by dialysis against cellulose membrane (MWCO-0.5 kDa) for 24 h (dialysate replacements for every 4-5 h) (yield 40%). The molecular weight and structural characterization of the polymers were performed by gel permeation chromatography and NMR spectroscopy respectively. The purified polymer had a molecular weight of 2300 (Mn) and poly dispersity (Mw / Mn) (1.2) was determined from gel permeation chromatography analysis. The dn / dc of HPOD polymer was 0.12 mL / g. The NMR structural analysis is shown in FIGs. 2 and 3, Scheme 1 and Table 1. In the1H NMR spectrum (FIG. 2) the protons of the polymer backbone were observed between 3-4 ppm, and the initiator, trimethylolpropane, proton peaks were observed around 0.5-1.5 ppm. A set of well-resolved peaks for different carbons were observed between 60-80 ppm in the13C NMR spectrum (FIG. 3). The monomer to initiator ratios and other conditions used for the synthesis of rest of the HPODs are given in Table 2.Table 1.13C NMR structural characterization of PG dendrimer-G3, HPG, and HPOD. The representative structural units are reported in Scheme 1. Integrated spectra were used to calculate DB.Table 2. Reaction parameters used to generate the library of HPODs.aCs(0H).H20 was used.bDioxane (3 mL) was used as an emulsifying agent.

[0153] For HPOD-1, simple precipitation protocols (MeOH / diethyl ether) were used to obtain the desired poly dispersity of the polymer and dialyzed for a short period of time to remove any organic solvents (MWCO-0.5 kDa, 8 h). For HPOD-2 and 3, after precipitation, dialysis purification was sufficient enough (MWCO-0.5 kDa) to obtain the polymers with desired molecular weight. For HPOD-4, after precipitation, either the dialysis or the fractional precipitation purification method was employed depending on the amount of lower molecular weight fractions in the crude reaction mixture. For HPOD-5, after precipitation, the fractional precipitation method was adopted by maintaining the constant volumes of methanol and varying the volumes of diethyl ether and finally dialyzed (MWCO-3 kDa) to remove any organic impurities. 'H NMR (400 MHz, D2O- 2): 5 0.83 (m, 3H), 1.29 (m, 12), 3.37 (m, 2H), 3.40-4.20 (m, 10H).13C NMR (75 MHz, D2O- 2): 61.0, 62.5, 69.0, 69.4, 71.6, 71.8, 72.4, 78.0, 79.6.Hydration studies of HPODs

[0154] Hydration of the HPODs was determined using differential scanning calorimetry (DSC). All the samples were dried at 40 °C under vacuum overnight to make sure that the water content was very minimal by thermogravimetric analysis (TGA). The dried polymer was used to prepare the polymer solution (7-10% WAV) in Milli-Q™ purified water, and approximately 20 pL was loaded into a Tzero aluminum hermetic sample pan and closed with an appropriate lid. The sample pan was cooled down to -20 °C and warmed to -5 °C at the rate of 2 °C / min.Heating of the sample was further continued from -5 °C to +5 °C at the rate of 0.2 °C / min and to +20 °C at the rate of 2 °C / min. The enthalpy of fusion of polymer solution or pure water was determined by integrating the area under the respective peak of the DSC trace. An empty pan was used as a reference. The same protocol was repeated for all HPODs, hyperbranched polyglycerol (HPG), polyglycerol dendrimer (PGD), and trehalose.

[0155] The number of water molecules bound per polymer was calculated using the following equation:[Nbw] = [AHfo (Wtw - WtP) -AHfps x Wtps] I [AHfoxMWmo x Np] where Nbw = number of water molecules bound per polymer; A H fps, AHfo= fusion enthalpy of polymer solution and pure water respectively; Wtw, Wtp, Wtps= weight of the pure water, pure polymer alone and polymer solution respectively; and Np = number of moles of polymer taken.

[0156] Since HPODs are highly hydrated, the correction factor for moisture content was applied to obtain the actual weight of the polymer based on the thermogravimetric analysis data. Each of the hydration measurements were repeated at least twice.Hydrodynamic size and diffusion coefficient measurements

[0157] Diffusion coefficient of different polymers was determined by pulse field gradient-nuclear magnetic resonance (PFG-NMR) experiments using a 400 MHZ spectrometer at 25°C, using D2O as a solvent (Anilkumar et al., 2020; Imran ul-haq et al., 2012; Price, 1997). The parameters (Tl-spin / lattice relaxation, Pi-90° pulse, 5-the width of a bipolar pair, and A-the diffusion time) of the experiment were optimized, and the data was acquired using a pulse program, Ledbpgp2s (from Bruker). The gradient strength (G) was altered from 1 to 32 G / cm in 16 steps (FIG. 4). Free induction decays were averaged over 8 scans using 4 dummy scans. The diffusion coefficient (Dt) was extracted (XWINNMR-3 software) using a non-linear square curve fitting to the relationship between pulse field gradient and echo attenuation, by the following equation. ln[A / Ao] = -Dt [y2G252(A- 5 / 3)]

[0158] Hydrodynamic radius ( / / >) was determined by Stoke-Einstein equation (Dt = I<BT / 6TH'| / I1) using the diffusion coefficient obtained from PFG-NMR experiments.Mass spectrometry characterization of HPG and HPOD

[0159] HPG and HPOD samples were characterized, following purification, using Matrix-Assisted Laser Desorption / Ionization-Time of Flight (MALDI-TOF) and Electrospray Ionization (ESI) mass spectrometry to determine the molecular weight of the sample and the repeating units. The samples were dissolved in Milli-Q water at 1 mg / mL and 2.7 mg / mL for MALDI-TOF and ESI, respectively. To prepare the matrix for MALDI-TOF mass spectrometry measurements, 2,5-dihydroxybenzoic acid (DHB) was dissolved in 50:50 acetonitrile: water solution at 10 mg / mL. 10 pL of the matrix was mixed with 1 pL of the sample and 1 pL of the sample / matrix mixture was deposited onto the MALDI target plate. The measurements were obtained on a Bruker Autoflex™ Speed LRF MALDI-TOF in positive ion linear mode. Prior to ESI mass spectrometry measurements, the sample was analyzed by a Hewlett Packard™ 1100 series High-performance liquid chromatography (HPLC) system using 100% acetonitrile as the mobile phase. Following HPLC, ESI mass spectrometry measurements were obtained on a Bruker HCT Ultra PTM in positive mode.Ice recrystallization inhibition studies

[0160] All the samples were prepared using 30 wt% sucrose solutions containing different polymers (HPOD-4, PVA (8 kDa) at 0.1, 1, and 10 mM and hydroxyethyl starch (HES; 180 kDa) at 0.1 and 1 mM). A droplet (0.5 pL) was added onto a microscope glass slide before pressing against another glass slide to create a film sealed by adding silicon grease along the edges. The space between two glass slides was kept consistent (aboutl60 pm thick) by using a small piece of aluminum foil as a spacer for all films prepared. The film was then placed onto a temperature-controlled flow cell coupled to an optical microscope (Axiolab; Zeiss™, Oberkochen, Germany) with a charge-coupled device (CCD) camera. The film was cooled to -50 °C to form small ice crystals (about 15 pm radii). Then, the film was heated to -9.1 °C, a temperature at which larger ice crystals grew at an appreciable rate at the expense of smaller ice crystals in the controls (i.e. no polymers). The temperature was held at this condition for 120 minutes. Digital images of the ice crystals were obtained using the optical microscope in transmission bright field mode every 10 minutes for 120 minutes. Images were analyzed using ImageJ software for crystal radius and reported as average ± standard deviation.(b) LNP cryopreservation studiesMaterials

[0161] CleanCap™ EGFP mRNA (5moU) was purchased from Cedarlane, mRNA encoding for firefly luciferase (fLuc) and saRNA (fLuc) were synthesized using MEGAscript T7 transcription kit from Invitrogen™ and was capped using the ScriptCap Capl capping system purchased from CELLSCRIPT, Cholesterol, 18:0 PC (DSPC) 1 ,2-distearoyl-sn- glycero-3-phosphocholine, powder, and DMG-PEG2K were obtained from Sigma- Aldrich, DLin-MC3-DMA was obtained from NanosoftPolymers (Salem, USA), NanoAssemblr™ Ignite Cartridges were purchased from Precision Nanosystems. Rediject D-Luciferin Bioluminescent Substrate was purchased from PerkinElmer™ (USA).LNP preparation and preservation

[0162] mRNA-LNPs encoding for enhanced green fluorescent protein (EGFP) were prepared on a NanoAssemblr (Precision NanoSystems Inc., Vancouver, BC, Canada) at a total volume of 1.5 mL with a N / P ratio of 3:1 and a 12 mL / min flow rate. The aqueous mRNA- containing buffer solution (pH=4) was rapidly mixed with the lipids (from Acuitas Therapeutics™, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3) / distearoylphosphatidylcholine (DSPC) / Cholesterol / PEG- Lipid 2000 at a ratio of 50 / 10 / 38.5 / 1.5) in ethanol and then diluted into phosphate buffered saline (PBS) and purified with Amicon™ 10k MWCO. Typically, the mRNA-LNP solutions were used at concentrations between 120-200 pg / mL for the preservation studies. Similarly, mRNA-LNPs and saRNA-LNPs encoding fLuc were prepared using the Pump 33 DDS Syringe Pump (Harvard Apparatus). The aqueous phase (mRNA (fLuc) or saRNA (fLuc) in 25mM sodium acetate buffer at pH 4.53) and lipid phase (made up of SM-102 / l,2-Dioleoyl-sn-glycero-3-PE (DOPE) / Cholesterol / DMG-PEG-2000 at a molar ratio of 45:17.5:36.25:1.25 in ethanol) were rapidly mixed) with a N / P ratio of 10 and 16 mL / min flow rate. The prepared LNPs were then diluted into PBS and purified with Amicon™ filters (10KMWCO) to a concentration around 150 - 200 pg / mL. The size and PDI were determined by dynamic light scattering (1: 100 dilution in PBS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) and the encapsulation efficiency (EE%) was determined using Quant-iT RiboGreen RNA Reagent and Kit (Thermo Fisher™) according to the manufactures protocol. To assess the integrity of encapsulated RNA, LNPs were subjected to lysis for RNA extraction, following established procedures (Kulkami et al., 2019). In brief, LNPs were combined with an equal volume of 2% Triton X lysis buffer andincubated on ice for 10 minutes to facilitate LNP lysis. Subsequently, RNA was isolated using the Monarch 10 pg RNA Cleanup Kit (New England Biolabs), adhering to the manufacturer’s recommendations. The eluted RNA was suspended in RNase-free water, and its concentration was adjusted to approximately about 250 ng / pL. RNA integrity was evaluated utilizing the Bioanalyzer RNA 6000 Nano Kit (Agilent™ Technologies), the 2100 Bioanalyzer Instrument (Agilent Technologies), and the Bioanalyzer 2100 Expert software (Agilent Technologies), following the manufacturer’s protocol. The assessment involved calculating RNA integrity based on the total peak area and the fdl transcript peak, as previously described (Ly et al., 2022; Casmil et al., 2023; Blakney et al., 2019).

[0163] In order to be considered freshly prepared as a positive control for the preservation experiments, the LNPs were used as soon as possible but always within a week after preparation. For preservation experiments, the mRNA-LNPs and saRNA-LNPs were mixed at a volume ratio of 1:1 with cry opreserving solution prepared in Tris buffer (tris(hydroxymethyl)aminomethan-HCl buffered solution, 20 mM, pH 7) at a final concentration of 20w / v of excipient, unless stated otherwise. Tris buffer only (20 mM, pH 7) was included as unpreserved reference that goes through a freeze-thaw-cycle or lyophilization process as well. Preserved and unpreserved tubes were stored at -70°C in a Mr. Frosty™ or lyophilized overnight. After thawing or reconstitution with Milli-Q water, respectively, the LNPs were characterized by size, PDI, RNA integrity, and EE% measurement, and then used for cell transfection of CHO and HeLa cells. Results were compared to the freshly prepared batch stored in the fridge that had never been frozen or lyophilized.(c) Cell culture

[0164] HeLa and CHO cells were cultured in Eagle’s Minimum Essential Medium (EMEM) and Roswell Park Memorial Institute (RPMI) medium, respectively, supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO2. For EGFP protein expression, cells were seeded at 50,000 cells / well in 48-well plates, cultured for Id and treated with 0.25 pg / well of mRNA. After 24 h of expression cells were dissociated with cell dissociation buffer, washed and subjected to analysis by flow cytometry (CytoFLEX™, Beckman Coulter). See FIG. 5 for gating strategy.(d) mRNA-LNPs and saRNA-LNPs expression in BALB / c mice

[0165] All animals were handled in accordance with the animal protocol (A21-0062) approved by the animal care committee, University of British Columbia. Female BALB / c mice aged 6-9 weeks were purchased from Jackson Laboratory (USA) and housed 3 per cage in a fully acclimatized room with constant access to sterile food and water ad libitum. Mice were imaged as previously described (Blakney et al., 2019). Briefly, mice were intramuscularly injected (both hind legs) with 5pg of mRNA (fLuc) LNPs (with or without excipients) or 1 pg of saRNA (fLuc) LNPs (with or without excipients) in a total volume of 50pL. On the day of imaging, mice were intraperitonially injected with 150 pL Rediject D- Luciferin Bioluminescent Substrate. 7 mins post injecting with the substrate, the mice were subjected to anesthesia (3-4% isoflurane) and imaged using the IVIS Spectrum In Vivo Imaging System (PerkinElmer) for 3 mins. Living Image analysis software by PerkinElmer was used to quantify the signal from the site of injection.(e) Protein stabilization studiesLysozyme stabilization assay

[0166] The lysozyme (Sigma- Aldrich) and Micrococcus luteus (Sigma- Aldrich) labelled with fluorescein isothiocyanate (FITC) were prepared at 100 U / mL and 1 mg / mL in water, respectively. The stabilizing agents, trehalose (Sigma Aldrich), HPODs, and other polymers (PEG, HPG, dextran, and poly(N,N-dimethyl acrylamide) (PDMA)) were prepared in reaction buffer (0.1 mg / mL, Dulbecco’s phosphate buffered saline (DPBS), pH 7.4) at 50 mg / mL (final concentration). Lysozyme solution (50 pL) was mixed with 50 pL of stabilizing agent in a 1.5 mL Eppendorf and heated at 60 °C for 30 minutes in a water bath or lysozyme alone with buffer, then cooled in an ice-bath for 15 minutes. The intact lysozyme activity was measured with the EnzChek™ Lysozyme Assay Kit (Thermo Fisher Scientific™). Micrococcus lysodeikticus cell walls (1 mg / mL, 100 pL) were mixed with the samples and incubated at 37 °C for 1 h in a light protected environment. The intact lysozyme lyses the bacterial cell wall and restores the fluorescence signal of FITC. Labelling of FITC with Micrococcus lysodeikticus cell walls causes the quenching of the fluorescent signal. The increase in fluorescent readings at 494 and 518 nm is proportional to the intact or activity of lysozyme. The data was normalized to the activity of the original activity of the lysozyme. All the analysis was performed on three independent experiments with two replicates.Insulin stabilization assay

[0167] Solutions of insulin (2 mg / mL) (Sigma- Aldrich) and all the stabilizing agents were prepared in glycine buffer (50 mM, pH = 2.5) to a final concentration of 0.5 mM, for all. The efficiency of the stabilizing agents was measured by a Thioflavin T fluorescence measurement assay (Mancini et al., 2012; Vilasi et al., 2008). Thioflavin T (ThT) was prepared in tris-buffer (10 mM, pH = 8.0) to a concentration of 20 pM. Insulin forms fibrils upon heating and these fibrils react with Thioflavin T which enhances the fluorescence. Insulin (100 pL) was mixed with 100 pL of stabilizing agent (or buffer) in a 2 mL glass vial and heated at 60 °C for 48 h. Fresh Thioflavin T solution was prepared before each fluorescence measurement. Thioflavin T (20 pM, 190 pL) was mixed with 10 pL of the heated samples, and centrifuged for 10 minutes. Then the samples were re-suspended using a pipette and fluorescence excitation and emission was measured at 450 and 486 nm respectively and is shown in FIG. 6. The data was normalized to the activity of the intact insulin that has never been heated. All the data are reported on three independent experiments (N = 3).[i-Galactosidase stabilization assay

[0168] The / / -galactosidase (Sigma Aldrich) solution (0.4 mg / mL) was prepared in sodium phosphate buffer (pH 7.0, 10 mM) (Xue et al., 2017). Three different concentrations of both stabilizing agents, HPOD-2 and trehalose (4 / 2 / 0.4 mg / mL), were prepared in sodium phosphate buffer (pH 7.0, 10 mM). G-Nitrophenyl- / / -galactoside (ONPG, Sigma- Aldrich) (1 mg / mL) was prepared in Milli-Q water and heated at 37 °C for 15 minutes to dissolve. / / -Galactosidase (25 pL) was mixed with 75 pL of stabilizing agent in a 1.5 mL Eppendorf™. All the samples were first frozen along with / / -galactosidase alone by immersion in liquid nitrogen until all liquid appeared frozen (approximately for 5 min). The samples were lyophilized for two 4 h cycles and one overnight cycle (15 h). After each cycle, 100 pL of Milli-Q water was added to each sample and again lyophilized. After the third cycle, 50 pL of Milli-Q water was added to all samples. ONPG solution (30 pL) was added to all samples and allowed to react in an environment devoid of light for 5 minutes. The intact / / -galactosidase cleaves the ONPG, generates a yellow compound (OD-420 nm). The reaction was then stopped with 50 pL of IM NazCOs solution and UV-Vis absorption was read at 420 nm.(f) Preservation of antibody activity

[0169] Two polymers (HPOD-2 and HPOD-4) and trehalose were used for preservation of antibody function after three repeated freeze and thaw cycles (for every 24 h) and the analysis was performed using flow cytometry throughout 3 days. Anti A antibody (cat no: ab2521 [Abeam™]) was mixed with each of trehalose (100 mg / mL), HPOD-4 (100 mg / mL), and HPOD-2 (100 mg / mL) in equal volumes and stored at -20 °C for further analysis. Antibody mixtures were then thawed daily (for three consecutive days) and incubated with 1% hematocrit A red blood cells (RBCs; 1 :500, RT, 30 min) that were collected after complete removal of platelet rich plasma (PRP) after centrifugation (Allegra X-22R centrifuge [Beckman Coulter™]) of whole blood at 1000 g for 5 min to assess the antibody function. For detection of successful preservation of antibody function, a secondary Alexa Fluor 488-labelled antibody (goat anti-mouse IgM (heavy chain) cross-adsorbed secondary antibody, Alexa Fluor™ 488: cat no. A21042 [Invitrogen™]) in a 1:300 concentration was applied at room temperature for 30 min and data were collected using CytExpert Software 2.3 (Beckman Coulter) on a CytoFLEX™ flow cytometer. Fresh antibody aliquot that never went through freeze and thaw cycles was used as a control. 100% RBC population (10000 counts) was used for flow cytometry assessment. To assure the purity of the cell population being assessed, naive RBCs were used to set a gate for the data collection. Data were collected only for the cell population defined by the gate. The following protocol was used for gating strategy. Naive RBCs at 1% hematocrit (were collected after centrifugation of whole blood at 1000g for 5 min for the complete removal of PRP) were used to set a gate for the data collection. To confirm the cell population being assessed is RBCs, naive RBC samples were treated with 1 / 100 R-phycoerythrin (RPE)-conjugated monoclonal mouse anti -Human CD235a, Glycophorin A / RPE, Clone JC159 (cat no: R7078 [Dako™]), at 4 °C for 30 min and assessed on a CytoFLEX flow cytometer [Beckman Coulter] to set a gate for the data collection. For 'positive' antigen A expressing samples, blood group A was used, and for 'negative' antigen A expressing samples, blood group O was used as controls. 'Control' blood samples were typed using Micro Typing System (MTS) cards [ID-Micro Typing SystemTM (ID-MTSTM) Gel Cards from Ortho Clinical Diagnostics™] according to the manufacturer’s protocol prior to the flow cytometry assessment.(g) Biocompatibility studies:Cytotoxicity analysis

[0170] Cell viability was assessed in HUVECs, EAHy, and BJ cells, using the (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT) assay. The cells were seeded at 10,000 cells / well on a 96 well flat bottom plate (Coming™) and allowed to settle and adhere for 24 h before addition of HPOD samples. HPOD samples (10 pL) were prepared in regular growth media (90 pL) to obtain final polymer concentrations of 1 mg / mL for the library screen and up top 40 mg / mL for HPOD-4. Cells were incubated with the HPODs for 48 h. After 48 h of incubation, samples were washed with PBS (1 x, pH 7.4) followed by addition of 100 pL of fresh media and 10 pL of 12 mM MTT reagent (ATCC™). After 4 h, 100 pL of sodium dodecyl sulphate (SDS)-HCl was added to solubilize the generated formazan. After solubilisation for 2 - 24 h, absorbance at 570 nm was read on a SpectraMax™ 190 microplate reader (Molecular Devices™) and compared to saline controls. Measurements were done in quadruplicates and the study was repeated three times. Average values and standard deviation are reported.Effect of HPOD on blood coagulation

[0171] Influence of HPODs on blood coagulation (blood compatibility) was evaluated using activated partial thromboplastin time (aPTT), prothrombin time (PT), platelet activation, and hemolysis assays following previously published protocols (Imran ul-haq et al., 2012; Abbina et al., 2019). Blood was collected from the donors with written consent at the Centre for Blood Research and followed the protocols according to the Clinical Research Ethics Board, The University of British Columbia. Blood was collected in 3.8% sodium citrated tube with a blood / anti coagulant ratio of 9:1 (BD Vacutainer™ Buffered Citrate Sodium (0.105 m; 9:1)) or ethylenediaminetetraacetic acid (EDTA; Fisher Scientific, New Jersey) or in serum tubes. Fresh blood was centrifuged at 150 x g for 15 min to isolate platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was prepared by centrifuging citrated whole blood samples at 1200 x g for 20 min. RBC suspensions were prepared by washing packed RBCs with PBS 3 times.Activated partial thromboplastin time (aPTT)

[0172] HPOD solutions at given concentrations (10x) were prepared in saline. Heparinized human platelet poor plasma (PPP) was prepared by adding unfractionated heparin (UFH) to sodium citrated human anticoagulated PPP to give a final heparinconcentration of 4 lU / mL. Heparinized PPP was used as a positive control. HPOD solution (25 pL) was mixed with 225 pL of citrated PPP (1:9 v / v HPOD:PPP) to generate the given concentration of HPOD. Then, 50 pL of the suspension and 50 pL of APTT reagent (Dade™ Actin™ FS Activated PTT, Siemens™ / Dade-Behring) were added to a cuvette with metal ball and incubated for 240 sec to reach a temperature of 37 °C. Next, 50 pL of 0.025 M CaCh was added to the mixture to initiate clotting, and clotting time was measured on a STart™4 coagulometer or STA Satellite Max (Diagnositca Stago, France). PBS was used as a negative control for the experiments. All experiments were performed in triplicates from three different donors and the average values ± s.d. are reported.Prothrombin time (PT)

[0173] HPOD solutions (10x) were prepared in saline. The stock solutions of the HPODs (25 pL) was diluted with sodium citrate anticoagulated PPP (225 pL; 1:9 v / v HPOD:PPP) to prepare the given concentration of HPOD. Next, 50 pL of this solution was added to a cuvette with a metal ball and incubated for 60 sec to reach a temperature of 37 °C. Afterward, 100 pL of synthetic thromboplastin reagent was added to initiate the coagulation cascade. All experiments were performed on a STart4 coagulometer or STA Satellite Max (Diagnostica Stago, France) in triplicates using three different donors and the average values ± s.d. were reported. PBS was used as a negative control. EDTA-treated PPP was used as a positive control.Platelet activation

[0174] The influence of the HPODs on platelet activation in platelet rich plasma (PRP) was measured by flow cytometry (BD FACS Canto III flow cytometer (Becton Dickinson). Briefly, 10 pL of HPOD (10x concentration) in PBS l x was mixed with 90 pL of PRP (1 :9 v / v HPOD: PRP) and incubated at 37 °C for 1 h. After 1 h incubation, the PRP / HPOD mixture (5 pL) was diluted with 50 pL of prepared antibody (2.5 pL anti-CD62P-PE antibody (Beckman Coulter) and 47.5 pL PBS). The solution was incubated for 20 min in the dark at RT, then further diluted with PBS (300 pL). The level of platelet activation was analyzed by flow cytometry, whereby 10,000 events were acquired in the platelet gate, which was determined by positive anti-CD42a-FITC fluorescence (> 99% positive). Duplicate measurements were performed using PRP from three separate donors, and the average ± s.d. values were reported. PBS, an PE conjugated goat anti-mouse IgG polyclonal isotype antibody, and thrombin receptor-activating protein (TRAP) activated platelets were used as negative, background, and positive controls, respectively.Red blood cell lysis

[0175] Hemolysis was assessed using Drabkin’s reagent by determining the concentration of released hemoglobin from lysed red blood cells. HPOD solutions (10x) were prepared in PBS and incubated with washed RBCs (10% hematocrit) for 1 h at 37 °C (1 :9 v / v polymerRBC suspension). RBCs incubated with distilled water (1: 15 v / v blood: water) was used as a positive control (100% lysis) and RBCs in PBS as negative control. The samples were mixed gently and 5 pL of suspension from each sample was transferred into a well containing 250 pL Drabkin’s reagent and 15 pL PBS. Each sample was then centrifuged at 1000 x g for 1 min, and 20 pL of supernatant was added to a separate well containing 250 pL of Drabkin’s reagent. The absorbance of hemoglobin (Hb) at 540 nm was measured on a SpectraMax M3 microplate reader (Molecular Devices) for both RBC suspension and supernatant.Percent lysis = (Hb concentration in supernatant / Hb concentration in suspension) x 100

[0176] The study was repeated with blood from 3 different donors and data was reported in average values ± standard deviation.(h) Mouse tolerance study:

[0177] The biocompatibility in vivo work was conducted at the BC Cancer Agency; (Animal Ethics # A22-0274). Female Balb / cAnNHsd mice were purchased from Envigo and acclimated 7 days prior to study start. Mice were caged in autoclaved Allentown™ ventilated caging at a capacity of 4 animals / cage during the course of the experiment. Cages were changed bi-weekly. The mice were injected with one dose intravenously at 200 pL and returned to their cages. Therefore, the mice were individually weighed and injected with amount (polymer) per body weight (mouse) using HPOD solutions at 500, 250, 100, and 50 mg / kg, a PBS injection was used as control. There were 4 mice in each group and two sets were injected for short term and long-term toxicity. They were continually monitored for acute signs of toxicity for two hours following each dose of test compound administration and clinical observations were monitored a minimum of once more at 6-8 hours, then daily monitoring afterwards. Upon termination after 1 day and 14 day to test for acute and chronic toxicity, major organs (heart, spleen, kidneys, lungs, and liver) were harvested and stored in10% normal buffered formalin. Blood samples were taken and the plasma stored at -80 °C until further analysis. The organs were sectioned by Waxit Inc. (5 pm thick slices) and stained (haematoxylin and eosin stain). Histopathological analysis was performed at Vancouver General Hospital by the group of Prof. Dr. Caigan Du. Serum samples were analyzed with ALT and AST activity assay kit (Sigma Aldrich), and with LDH assay kit (abeam).(i) Statistical analysis

[0178] Statistical analysis was performed using GraphPad Prism 9 (Graphpad Software, San Diego, USA). Pairwise multiple comparisons were conducted using unpaired t-test or ANOVA. Unless otherwise stated, all experiments are represented as average ± standard deviation of a minimum of 3 independent studies performed on different days.II. Results(a) Design and Development of Semi-dendritic Highly Hydrated Polymers

[0179] To achieve a highly hydrated structure for the preservation of biotherapeutics, the design strategy involved the combination of a hydrophilic poly ether-poly ol based backbone, with an optimized topology that facilitates the accommodation of more unfreezable water. Considering the fact that extensive hydrogen-bonding networks and the presence of multiple water-binding functional groups may provide highly hydrated macromolecules, a monomer 3-(oxiran-2- ylmethoxy)propane-l,2-diol (OPD) (glycerol glycidol monoether) (Scheme 2) was developed. This monomer can be polymerized through ring opening multi-branching polymerization (ROMBP) to create a dendritic poly ether backbone with numerous hydroxyl groups.3-(allyloxy)propane-l,2-diol OPDScheme 2. A chemical scheme showing the synthesis of monomer (3-(oxiran-2-ylmethoxy) propane- 1,2-diol) (OPD).

[0180] A branched polymer structure was chosen due to its potential advantages, including limited inter-chain interaction, low intrinsic viscosity, and high biocompatibility as opposed to the linear structure (Abbina et al., 2017; Kainthan et al., 2006). The monomer was synthesized by epoxidation of 3-(allyloxy)propane-l,2-diol in decent yields (60-70%) andcharacterized by different spectroscopic techniques. Together with1H.13C, and HSQC NMR (FIG. 1) and mass spectrometry, the structure of the OPD monomer was confirmed.

[0181] By employing anionic ROMBP (Sunder et al., 1999), a small library of hyperbranched poly (3-(oxiran-2-ylmethoxy) propane- 1,2-diol (HPOD) polymers were synthesized with molecular weights ranging from 640 to 11,500 Da (see FIGs. 2, 3, 7 and 8) and Table 3 by changing the monomer to initiator ratios, varying base and using emulsification agents. The hydrodynamic size (2Rh) of the HPODs was measured by the Stokes-Einstein equation, Rh = kBT / 67iqDt; the translational diffusion coefficient (Dt) was determined using pulse-field gradient nuclear magnetic spectroscopy. A key challenge faced was the dehydration of the monomer for polymerization. A considerable amount of bound water was found to be associated with OPD monomer (7-18 wt% water based on thermogravimetry analysis), which necessitates very stringent dehydration protocols before attempting ROMBP. The obtained honey -like HPOD polymers were highly hygroscopic and water-soluble.Table 3. Characterization of HPODs. Absolute molecular weight and intrinsic viscosity of the HPODs were determined by triple detection gel permeation chromatography analysis (multi-angle static light scattering (MALS), refractive index detector, and viscometer detectors). HPODs are abundant with bound water, and the hydration was determined by measuring the fusion enthalpy of polymer solution by differential scanning calorimetry.aLee et al., 2022.

[0182] The structural and physical characterization of HPOD polymers were performed using multiple analytical methods. The absolute molecular weights of the HPODs ranged from 640 to 11,500 Da (Table 3 and FIG. 8) obtained by GPC-MALS. The hydrodynamic sizes of HPODs were in the range of 2 to 4.8 nm as measured by pulse field gradient NMR spectroscopy, and as expected there was a modest increase in the hydrodynamic size with the increase in molecular weight (Table 3, Table 4, and FIG. 4). HPODs also showed low intrinsic viscosities(rf) in water in the range of 4.2-6.5 mL / g (Table 3), which is low compared to linear polymers, such as PEG, polyvinylpyrrolidone (PVP) or PVA but similar to that of globular proteins (Armstrong et al., 2004). There is a slight increase in intrinsic viscosities of HPODs (Table 3) with molecular weight. NMR (1H,13C, and DEPT), MALDI-ToF, and ESI-MS and were used for the structural characterization of the HPODs (FIGs. 9 and 10 and Table 5).Table 4. Diffusion coefficient of HPODs was determined using pulse field gradient-nuclear magnetic resonance (PFG-NMR) experiment (400 MHz, D2O, 25°C). A group of peaks at 5 4.06, 3.99, 3.87, 3.77, 3.69, 3.60, and 3.41 ofHPOD spectra were used to determine the diffusion coefficient.Table 5. The ratio between the different branching units of the HPODs shown in FIG. 9 was used to calculate their degree of branching (DB) which was calculated using the equation, DB = (2D±sD) / (2D±(4 / 3)sD±2 / 3(L)) (Sunder et al., 1999).

[0183] In the1H NMR, the protons of the polymer backbone were observed between 3-4 ppm, and the initiator, trimethylolpropane, proton peaks were observed at 1-2 ppm (FIG. 2). A set of well-resolved peaks for different carbons were observed between 60-80 ppm on13C NMR spectrum (FIG. 3). To elucidate additional structural information of HPOD polymers, the degree of branching (DB) of HPODs was measured using inverse-gated13C NMR (FIG. 9). As it is an AB3 monomer, DB was calculated using the equation, DB =2D+5D / (2D+(4 / 3)SD+2 / 3L)) (Sunder et al., 1999). Four structural configurations of HPODs were labeled as perfect dendritic (D), semi dendritic (sD). linear (L), and terminal units (T). The possible structural linkages in these configurations were described as D13-D17-D18 in dendritic, L13, L17, and Lis in linear configuration, and SD13-SD17, sD -sDis in semi dendritic units and their intensities were analyzed using13C inverse-gated and DEPT NMRs (FIG. 9). Although different proportions of these structural configurations (D, s D. L and T) are theoretically expected, the proportions at the given NMR experimental conditions could not be identified and thus, the theoretical approximations to derive the DB of the polymers were adopted. The DB increased with the molecular weight of the polymer; HPOD-1 (Mn-640 Da) had 23% branching and DB reached up to 46% for HPOD-5 (Mn-11,500 Da) (Table 5).

[0184] Additional structural information was obtained from MALDI-ToF mass spectrometry (Kosyakov et al., 2019) including the monomer incorporation, presence of initiator unit, and the incorporation of counter ions. The MALDI-ToF spectra of HPOD-1 and HPOD-2 are given in FIGs. 10-12. The mass difference between the peaks represents precisely the molecular mass of OPD monomer (148 m / z) in both spectra suggesting that OPD monomer is incorporated within the polymer and the mass of the repeating unit is 148 Da. The MALDI spectra reveal additional information. Each of the peaks was made up of either H+ or Na+ as a cation counter ion adduct and each peak was made up of the sum of the mass of the core unit l,l,l-tris(hydroxymethyl)propane (134 m / z) (see Table 6 for the analyses of different peaks). An intriguing observation is that each peak of HPOD in the spectra has 2 to 4 bound water molecules depending on the m / z value of the peak. For example, in the case of HPOD-2, higher m / z peaks do have 4 water molecules in comparison to peaks at lower m / z values. Detailed analyses of each peak of MALDI spectra for both polymers, and the calculation is given in FIG. 11 and FIG. 12 and Table 6.Table 6. MALDI-ToF data analysis of selected peaks of HPOD-2, HPOD-1, and HPG. The peaks are each spaced by their respective monomer unit and have peaks for different counter ions. However, water adducts can only be found in each HPOD spectrum and no water adducts are seen for HPG.Peak constituents (Mw)„ . .TNo. and Theoretical MWExperi .ment .a .l . Count+er No. and MW ,i> M . .w .. .Polymer _ , _r, .] Initiator’ ion from PeakJPeak MW [m / z ( 'H+or ( 'OPD units)7" ( .wat ,er .) ." const .i.t.uent .sNa 1 Im / Z][m / z] [m / z]781.1 134 1 4 x 148= 592 3 x 18 = 54 781763.1 134 1 4 x 148= 592 2 x 18= 36 763HPOD- 1225.9 134 1 7 x 148=1036 3 x 18 = 54 12252 1266.0 134 23 7 x 148= 1036 4 x 18 = 72 12651117.8 134 23 6 x 148= 888 4 x 18 = 72 1117614.7 134 1 3 x 148= 444 2x 18 = 36 615HPOD- 632.8 134 1 3 x 148= 444 3x 18 = 54 6331 781.1 134 1 4 x 148= 592 3x 18 = 54 781929.4 134 1 5 x 148= 740 3x 18 = 54 929506.2 134 1 5 x 74d0 506526.5 134 23 5 x 74 0 1193600.7 134 23 6 x 74 0 601674.9 134 23 7 x 74 0 675823.2 134 23 9 x 74 0 8231193.8 134 23 12 x 74 0 1193“molecular weight of the initiator, l,l,l-tris(hydroxymethyl)propane - 134 Dabmolecular weight of the monomer, 3-(oxiran-2-ylmethoxy)propane-l,2-diol (OPD) - 148 Dacmolecular weight of water - 18 Dadmolecular weight of glycidol- 74 Da

[0185] Similar water adducts were reported for the MALDI spectra of sugar molecules (Kosyakov et al., 2019), suggesting that HPOD polymers are highly hydrated as hypothesized and are quite different from other branched poly ether polyols (Wilms et al., 2007). In addition, ESI mass spectra of HPOD (FIG. 13) showed monomer and initiator incorporation, differences in repeating units and hydration. These features differentiate the HPOD structure from the well-studied hyperbranched polyglycerol (HPG) (Abbina et al., 2017; Sunder et al., 1999), which are prepared by anionic ROMBP of glycidol, an AEh type monomer and poly glycerol dendrimer (PGD) (Winther et al., 2012) (discussed in a later section).(b) Hydration characteristics of the HPODs

[0186] Important structural features of HPODs are their branched poly ether structure with significant linear structure content along with dendritic units with low degree of branching, and with an abundance of both primary and secondary hydroxyl groups. Together these properties are anticipated to generate a highly hydrating structure. Thus, the hydration characteristics of HPODs were investigated. The number of bound unfrozen water molecules associated with HPOD was determined from the hydration enthalpy of aqueous solutions by differential scanning calorimetry (FIG. 14) and results are displayed per monomer in Table 3 (Du et al., 2014). The hydration levels varied depending on the molecular weight of the HPODs, with hydration numbers ranging from 15 to 27 water molecules per monomer unit compared to about 22 for trehalose (Winther et al., 2012) and about 4 for PEG (Wolde-Kidan et al., 2021). Interestingly, lower molecular weights provided higher hydration values, which is, while notwishing to be limited by theory, most likely attributed to their less branched nature and thus more accessible hydroxyl groups throughout the polymer structure. When compared to trehalose, a disaccharide, which is the benchmark in the field, HPOD-1 and HPOD-2 demonstrated even greater amounts of water per monomer unit (Winther et al, 2012).(c) Cryopreservation of RNA-LNPs in the presence of HPOD excipients

[0187] Due to their highly hydrated nature, it was hypothesized that HPOD might be an excellent candidate to be used for cryoprotecting RNA-LNP formulations. Thus, mRNA encoding for enhanced green fluorescent protein (EGFP) was encapsulated into LNPs (mRNA-LNP) and a series of formulations were prepared by adding different HPODs as excipient. These were compared to an unpreserved control in buffer. To emulate the cold- storage condition of mRNA-LNP formulations, the formulations were exposed to a freezethaw cycle at -70 °C and the physicochemical properties were determined after thawing the next day. These were then compared to those of freshly prepared mRNA-LNPs that never went through a freeze-thaw cycle. mRNA-LNPs were considered as fresh if they were used within one week and their size, PDI, EE% and % transfected is referred to as original. Initial screening of the HPOD library at different concentrations (see FIGs. 15-18) was performed to identify the best preservation conditions and this was compared to FDA approved excipients sucrose and trehalose at the same concentration. Initially, the size and PDI was looked at by DLS, and the encapsulation efficiency (EE%) was determined by the Ribogreen assay (see FIGs. 19-21). A small increase in size and PDI was seen when compared to the original values of mRNA-LNPs that never went through a freeze-thaw cycle; the size increase was least pronounced for HPOD-2 and HPOD-5, meaning these polymers performed as well as FDA-approved sucrose formulations. Interestingly, an increase in size and PDI was observed prior to freezing (see FIG. 22). Thus, while not wishing to be limited by theory, it is more likely attributed to the additives interacting with the LNP corona, thereby increasing the hydrodynamic radius and not by altering the actual LNP structure. As depicted in FIG. 21 and FIG. 23, the increase in size is not associated with any loss in EE% or transfectability for all HPODs suggesting that all mRNA molecules were still encapsulated within the LNPs after going through a freeze-thaw cycle. This was confirmed by determining the RNA integrity after a freeze-thaw cycle, showing that HPOD as excipient was able to preserve the RNA (FIG. 24).

[0188] Next, cryo-preserved LNPs were used to transfect both CHO and HeLa cells (see FIG. 23 and FIG. 25). The cells were transfected for 24 h and then subjected to analysis byflow cytometry; values are compared to freshly prepared mRNA-LNP. Both high and low molecular weight polymers (HPOD-5 and HPOD-2) maintained the transfectability. The data showed superior performance to HPOD-4 and trehalose and were equally effective as sucrose at high concentration (Mitchell et al., 2016). The number of freeze-thaw cycles should be minimized to ensure the stability and integrity of formulations. However, it is desirable for an excipient to provide protection to the formulation for more than a single cycle. Therefore, a study was carried out wherein the formulation underwent three complete freeze-thaw cycles. Notably, despite the exposure to multiple cycles, the formulations demonstrated consistent and sustained transfectability (see FIG. 26). Additionally, confocal microscopy images of transfected HeLa cells clearly demonstrate that mRNA-LNP complexes cryopreserved with HPOD-5 exhibit consistent and uniform transfection, as evident by sustained EGFP expression both pre-freezing and post-freezing (see FIG. 27). Conversely, unpreserved mRNA-LNPs exhibited a significant decline in transfection efficiency.

[0189] HPOD-5 was then proceeded with and its in vivo transfection capabilities following intramuscular injection (z.m.) were evaluated. Thus, mRNA encoding for firefly luciferase (fLuc) encapsulated into LNPs was prepared. After storage at -70 °C both the unpreserved and cryopreserved formulations were injected into Balb / c mice and compared with freshly prepared mRNA-LNPs as reference (see FIG. 28). Luminescence was recorded on Day 2 and results are shown in FIG. 29. The HPOD-5 preserved LNPs showed no significant difference in luminescence compared to freshly prepared LNPs, meaning that no loss of transfectability was observed during the freeze-thaw cycle.

[0190] Further, the preservation of self-amplifying RNA based LNPs (saRNA-LNP) was evaluated. Unlike traditional mRNA, which only carries the code for one protein, saRNA contains the code for both the target protein and an RNA replicase enzyme, which can create multiple copies of the mRNA within the cell (Ko & Maynard, 2018). This leads to a much higher level of protein production, making saRNA a potentially powerful tool for gene therapy. The much longer chain length of saRNAs (about 10,000 nt) compared to mRNAs (about 1,000 nt) makes them much more prone to degradation and thus, cold- storage and careful preservation is of utmost importance. To demonstrate the scope of applicability the preservation of saRNA-LNP encoding for fLuc was investigated. After confirming the preservation of the physicochemical properties by HPOD in the case of saRNA-LNPs (see FIG. 30), studies of both preserved and unpreserved saRNA-LNPs inBalb / c mice were performed. FIG. 31 shows the results imaged at Day 7 post injection. Again, HPOD-5 fully preserved the transfectability with no significant difference to freshly prepared saRNA-LNP. With the additional advantages of low osmolarity (5.3 ± 2 mOsm for HPOD-4 versus 31.7 ± 2 mOsm for sucrose, at 10 mg / mL), being highly biocompatible (as discussed later), and having synthetic control, the highly hydrated excipient demonstrated its potential as a new cryoprotectant for LNP-based gene delivery.(d) Lyophilization of mRNA-LNPs

[0191] A small HPOD-library was screened for their efficacy as lyoprotectants and the high molecular weight HPOD-5 was identified as the best candidate based on size preservation and EE%. As shown in FIGs. 32-36, HPOD-5 worked as an excellent lyo-protectant for mRNA- LNPs. The mRNA-LNPs were lyophilized overnight, reconstituted in water, and size, PDI, EE% and transfection efficiency in HeLa cells were determined and compared to the values of freshly prepared mRNA-LNP (see FIGs. 32-35 and CHO cell transfection in FIG. 25). In all cases a significant size increase was observed when compared to the freshly prepared mRNA- LNP. This is a known effect in literature and, while not wishing to be limited by theory, thought to be attributed to a reorganization of the lipids upon reconstitution in water, however, the process is still not fully understood (Muramatsu et al., 2022; Ball et al., 2017). Regardless of the size increase, the PDI, EE%, and transfection in both HeLa and CHO cells are fully preserved by HPOD-5, to a similar or better extent than sucrose.

[0192] The in vivo performance of lyophilized formulations was then studied, and the results are shown in FIG. 36. The luminescence after i.m. injection of mRNA-LNPs was lower after lyophilization for all samples compared to the signal of freshly prepared mRNA-LNPs. The signal for HPOD-5 preserved mRNA-LNPs was as high as that of sucrose and the results show that the preserved LNPs had sufficient functional mRNA present. In order to investigate if there is a synergistic or additive effect of sucrose and HPOD one formulation with a 1 : 1 mix of sucrose and polymer was prepared whereby the final excipient concentration remained constant. The measured luminescence was slightly better however not significant when compared to sucrose or HPOD alone, indicating that the sugar and polymer may preserve LNPs in a cooperative fashion. Further studies may elucidate if the advantages gained from dry storage conditions would make up for the loss in transfection efficacy. Additionally, there are more factors to consider when lyophilizing, such as the optimization of the reconstitution buffer, storage of the lyophilized product, or the lyophilization method. To fully exploit thepotential of our material, further investigations are useful to evaluate its long-term stability under various storage conditions, including room temperature, -20 °C, and -70 °C.(e) Protein stabilization and excipient activity of HPODs

[0193] Next whether the highly hydrated HPODs can preserve other biotherapeutics such as proteins which are often very sensitive to different external stresses, and are prone to chemical and physical degradation was investigated (Mitragotri et al., 2014; Best, 2015). Four industrially and therapeutically relevant proteins were chosen for this study; lysozyme, insulin, / / -galactosidase, and an anti-blood group A antibody. The activities of these proteins after exposing them to external stresses such as freezing, heat, and lyophilization FIGs. 6 and 37-42) in comparison to the current industry standards were measured. The library screening studies for the stabilization of lysozyme from heat shock (60 °C for 30 min) showed the activity of lysozyme after being stabilized with different HPODs in comparison to trehalose as a GRAS listed excipient (see FIG. 37) at an identical concentration (50 mg / mL). HPOD-4 and HPOD-3 preserved the activity almost similar to the native protein demonstrating its excellent stabilization ability in comparison to best-known stabilizer trehalose. The low molecular weight HPOD-1 was not as effective as the other HPODs. Further, HPODs showed superior stabilization ability in comparison to different hydrophilic polymers, including PEG, PDMA, polyglycerol, and dextran (FIG. 42). The concentration-dependent activity for HPOD was then investigated, whereby HPOD-4 exhibited superior performance in comparison to trehalose (0.1, 0.5, 1, and 50 mg / mL) (FIG. 38). HPOD-4 is effective even at 0.5 mg / mL whereas trehalose did not show any concentration dependency.

[0194] The stabilization activity of HPODs against aggregation of insulin upon heating in comparison to other established excipients was next investigated (FIG. 6 and FIG. 39). Insulin was heated at 60 °C for 48 h in the presence of different stabilization agents (final concentration was 0.5 mM) and the amount / proportion of intact insulin was measured using a Thioflavin T based fluorescence assay. HPODs showed significantly better performance in inhibiting fibril formation compared to trehalose, PEG, and proline; no molecular weight dependence was observed for HPODs. Finally, the stabilization of proteins upon lyophilization and freeze-thaw induced stress was investigated as it is known to damage the structure of the proteins severely. / / -Galactosidase is an enzyme with significant industrial relevance, however, it denatures upon lyophilization (Bhatnagar et al., 2007). The activity of / / -galactosidase after exposure to multiple lyophilization cycles in the presence andabsence of HPOD was measured. Trehalose was used as a control (FIG. 40) (Chapanian et al., 2012). Both HPOD and trehalose were effective in stabilization of / i-galactosidase against lyophilization stress. However, HPOD-2 showed improved performance at low concentrations compared to trehalose. With this, it was of further interest to in evaluate the efficiency of HPODs for preserving the activity of antibodies upon repeated freeze-thaw cycles. Antibodies are usually stored at -20 °C; however, the retention of therapeutic activity of the antibodies after repeated thawing is a significant challenge in both academic as well as in industrial settings. An anti-blood group A antibody (anti-A antibody) was chosen as a model antibody and its activity for binding human blood group A (monofucosyl and difucosyl A antigens) on red blood cells (RBCs) after three repeated freeze-thaw cycles (-20 °C to RT, each cycle is repeated every 24 h) was measured using flow cytometry. A fresh antibody aliquot that never went through freeze-thaw cycles was used as a control (FIG. 41). HPOD-2 and HPOD-4 demonstrated the preservation of antibody function and exhibited superior performance compared to trehalose on day 1 and 2. While no statistical difference was observed among the stabilizing agents on day 3, it was evident that HPOD- 2 exhibited activity similar to that of the fresh antibody.

[0195] Collectively, these findings demonstrate that HPODs are promising stabilizing excipients for both industrially relevant and therapeutic proteins. HPODs effectively protected these sensitive agents against various external stresses commonly encountered in the respective applications.(f) Biocompatibility of HPODs

[0196] In contrast to natural polymers or small molecules, synthetic polymers are tunable, can tolerate a wide range of temperatures, are durable, and resistant to harsh conditions. Often their main limitation is lack of biocompatibility, especially when used at high concentrations. The findings described herein indicate that higher concentrations of HPOD generally performed better in preserving biological activity across most tested applications. As the process of removing excipients prior to utilizing a biotherapeutic is tedious and not a sustainable practice in most applications, a comprehensive evaluation of in vitro toxicity and in vivo tolerance of the HPOD library was performed. Cell viability, blood clotting, platelet activation, andRBC lysis was looked at for the different molecular weights and at high concentrations (see FIGs. 43-53). The testing revealed no influence on blood clotting using coagulation assays aPTT and PT, no hemolysis, andno platelet activation under all tested conditions. Incubation with endothelial and fibroblast cells did not lead to a decrease in cell viability for any of the tested conditions.

[0197] After seeing good hemocompatibility and in vitro tolerance, the toxicity of HPOD was evaluated in mice where a single escalating dose (up to 500 mg / kg) of HPOD was injected intravenously. The dose range was selected based on the fact that the highest dose injected was approximately 10-times lower than the maximum that will be injected in the case of a cryopreserved therapeutic. Blood samples and organs were assessed after 1 d and 14 d to test for acute and chronic toxicity and compared to saline injected controls (FIGs. 54-59). The levels of alanine aminotransferase (ALT) and aspartate amino transferase (AST) were examined to detect any indications of liver damage. Serum levels were not elevated compared to the saline control at either time point. In addition, no increase in lactate dehydrogenase (LDH) levels was found, suggesting no tissue damage. There were no significant changes in body weights of the mice after the injection in comparison to saline control (FIG. 58). Finally, major organs (referring to FIG. 56: lung 10, heart 12, kidney 14, liver 16 and spleen 18) were harvested 14 d after HPOD injection with the highest concentration and examined histologically. Tissue sections were stained using hematoxylin and eosin (H&E) and examined for tissue damage in a double-blind study (see FIG. 59). Pathological examination found no abnormalities in comparison to the control mice injected with saline. Taken together, these data prove that HPODs are well tolerated both in vitro and in vivo. This indicates that HPODs are ideal candidates to be used as excipients in the context of stabilizing biotherapeutics against heat, freezing, and lyophilization and may not have to be removed before administration.(g) The influence of architecture, topology, and hydration of HPODs

[0198] Having investigated the application of HPODs in the preservation of different types of biotherapeutics, their structural features that provide high hydration and preservation properties was further looked into. More specifically, the branching and branching-dependent hydration features of HPODs was investigated. The hydration characteristics of hyperbranched polyglycerol (HPG) and polyglycerol dendrimer (PGD) generation 3, two representative polyether polyols with different branching characteristics were also compared (Abbina et al., 2017; Haag et al., 2000).

[0199] The hydration of HPOD polymers was clearly dependent on the molecular weight and branching of the polymers. The low molecular weight polymers have more bound water molecules compared to the high molecular weight HPODs (FIG. 60). The degree of branchingand thus, the topology of HPOD, also influences the hydration properties (FIG. 61). A steep increase in the hydration was observed when the degree of branching decreased from about 46% to 23%. Above and below this limit, the bound water molecules per monomer unit seems to be getting saturated. Particularly, the significant presence of linear units and the extended dendritic units (Scheme 1) in HPOD offer a more open branching structure and space to hold a higher number of water molecules compared to polymers with higher branching.

[0200] Based on the branching- and MW-dependent hydration properties of HPOD, the hydration of those similar polyether-polyol families of dendritic polymers with different degrees of branching was investigated. It was anticipated that this would provide additional evidence on the influence of polymer topology on hydration. The hydration properties of HPOD (DB 40%), hyperbranched polyglycerol (HPG) (DB 57%) (Sunder et al., 1999) and poly glycerol dendrimer (PGD) (Zi eringer et al., 2012) (DB 100%) of similar molecular weight (FIG. 62 and Table 7) were compared. These polymers have a similar chemical composition but are topologically different due to the differences in branching structure (FIG. 62 and Scheme 1). Since similar molecular weight polymers were used, any change in the number of bound water molecules could only be attributed to the topological differences of the polymer. HPOD was found to have higher hydration (about 247 bound water molecules per polymer) compared to HPG (209 bound water molecules) and PGD with 140 bound water molecules (see Table 7 and FIG. 14 (lower)). The number of water molecules associated with these polymers increases as the degree of branching (DB) decreases. While not wishing to be limited by theory, this increase was attributed to the additional non-freezable water molecules accommodated in the space created by the linear extensions within HPOD compared to HPG or PGD, as well as the greater availability of hydroxyl groups within the polymers. The larger hydrodynamic size of HPOD supports the presence of additional space within the polymer structure.Table 7. Comparison of hydration of HPODs with polyglycerol polymers. Hydration of the macromolecules was influenced by DB. HPOD was found with more bound water molecules than HPG or PGD, demonstrating the influence of polymer topology. The hydrodynamic size (2 / i>) of the HPOD was measured by the Stokes-Einstein equation, / i> = kBT / 6?iqDt; Dt was determined using pulse- field gradient nuclear magnetic spectroscopy.Polymer Mn[Da] DB [%] Diffusion coefficient Hydrodynamic Hydration number

[0201] HPOD exhibits approximately 76% more bound water molecules than PGD of similar molecular weight; PGD has a more compact structure with hydroxyl groups located only on the outer periphery. Additionally, HPOD demonstrates higher hydration levels than the disaccharide trehalose, manifesting the outstanding hydration characteristic of HPOD (FIG. 14 (lower); about 44 water molecules / molecule of trehalose (378 amu) and 176 water molecules per polymer if the molecular weight is extrapolated to that of HPOD (1500 Da)) (Kawai et al., 1992). The higher hydration of HPOD is further supported by its slower diffusion rate and larger hydrodynamic size compared to HPG or PGD (Table 7). Furthermore, MALDI-ToF spectra of HPG, HPOD-1, and HPOD-2 are given in FIGs. 11 and 12 and the ESI spectrum of HPOD in FIG. 13. The MALDI-TOF spectra of both HPODs reveal not only the expected mass peak distribution indicative of its repeating units, but also the presence of adduct peaks. These adduct peaks correspond to the HPOD molecule complexed with water molecules (18 m / z), with multiplicities of 2, 3, or 4 water molecules observed, as detailed in Table 6. This finding is similar to that reported in the case of sugar molecules (Kosyakov et al., 2019). However, HPG and PGD (Winther et al., 2012) do not show any water adducts. The MALDI-ToF data supports the unique hydration behavior of HPOD, which is unlike other polyether polyols of similar chemical composition. Together these data support the claim that the structure and topology of these polymers are influencing the hydration characteristics even though these polymers have similar molecular weights, number of hydroxyl groups, and a poly ether polyol backbone.

[0202] In an attempt to correlate the hydration features to the cryoprotection ability of HPODs, their ice recrystallization inhibition efficiency was looked at in comparison to hydroxyethyl starch (HES) and polyvinyl alcohol (PVA) (Budke et al., 2014; Deller et al., 2014). Ice recrystallization refers to the growth of large crystals at the expense of small crystals, a process that can disrupt cells, protein structures, or soft particles like LNP during a freeze-thaw cycle. The ice recrystallization inhibition efficiency of these agents was determined by rapidly freezing sucrose-water solutions containing different polymers (HPOD, PVA, and HES) at about -50 °C to form small ice crystals (about 15 pm radii), and then annealing the samples at about -9 °C and monitoring the growth of large ice crystals (measurements of the diameter of the crystals in FIG. 64) at the expense of small ice crystals during the annealing process using cryomicroscopy (FIG. 63). While the concentration of HPOD-4 showed a significant influence on the growth of large ice crystals (see FIGs. 65and 66), the recrystallization inhibition was not different from sucrose and HES, indicated by non-significant differences in ice crystal diameters (see FIG. 64) (Deller et al., 2014; Mitchell et al., 2016). Based on the current data, HPOD and HES in sucrose solution exhibited less ice recrystallization inhibition compared to PVA. However, these substances demonstrated their effectiveness in cryopreservation, suggesting the presence of an alternative mechanism, while not wishing to be limited by theory, possibly involving the formation of a hydrated matrix around LNPs or proteins (Mitchell et al., 2016).III. Discussion

[0203] Preserving the activity of biotherapeutics is a crucial aspect of their development, as it greatly influences their pharmaceutical applicability. The importance of cryoprotective excipients became evident through their utilization in the approved mRNA-vaccines Comimaty (BioNTech / Pfizer) and Spikevax (Modema) during the COVID-19 pandemic (Strickley & Lambert, 2021; Brader et al., 2021). Given the anticipated growth of research in the field of LNP technology, it is important to address compatibility challenges and enhance stabilization methods to fully realize their potential. Exploring the avenue of lyophilization offers a promising opportunity for further investigation and advancement. Developing excipients for LNP protection poses several challenges due to the complex nature of the particles and their predisposition to various degradation pathways, such as the denaturation of the cargo, particle aggregation, or bursting. Excipients need to stabilize the RNA-LNPs and prevent or minimize these degradation processes during storage and administration, while at the same not interacting adversely with the RNA-LNPs in a way that would lead to structural changes or decreased activity (Brader et al., 2021). Compatibility issues can arise due to pH, ionic strength, or specific interactions between excipients and particles (Patel et al., 2021). Desirable excipients allow for formulation flexibility, including various dosage forms such as liquid solutions or lyophilized powders.

[0204] Similar challenges are faced in the development of excipients for proteins because of their susceptibility to denaturation, aggregation, oxidation, and proteolysis (Chennamsetty et al., 2009; Leader et al., 2008; Ko & Maynard, 2018; Mancini et al., 2012). There is a high demand for improved cryoprotectants, where hemotoxicity, immunogenicity, cytotoxicity, synthetic challenges, and difficulty in the generation of pharmaceutical grade compounds prevent the advancements in this field (Chennamsetty et al., 2009). Approved excipients include sucrose in antibody formulations (e.g. KEYTRUDA™ (pembrolizumab)), PEG and sorbitol are used inOgivri™, and human serum albumin is used in Blincyto™ (Strickley & Lambert, 2021). Further, trehalose is used as an excipient in many commercially available therapeutic products, including Herceptin™, Avastin™, Lucentis™, and Advate™ (Kawai et al., 1992); Kaushik & Bhat, 2003). Those excipients used, however, are not applicable to diverse pharmaceutical products and each application necessitates the evaluation and identification of suitable excipients (Kerwin, 2008; Shiraishi & Yokoyama, 2019; Best, 2015; Bhatnagar et al., 2007).

[0205] Herein, a new class of highly biocompatible branched polymer, (hyperbranched poly(3-(oxiran-2-ylmethoxy) propane- 1,2-diol) (HPOD)) is described, with exceptional hydration properties and its excellent excipient properties showcased. HPODs exhibited a unique capability to effectively preserve both mRNA- and saRNA-LNPs, stabilize them against degradation (see, for example: FIGs. 19-21, 23, 24, 27-29 and 31-36), and protect a spectrum of protein therapeutics (see, for example: FIGs. 37-41), safeguarding them against various stressors such as freeze-thaw cycles, lyophilization, and heat. Even though there was no consistent molecular weight to function relationship, HPODs as one class of polymer did preserve a variety of pharmaceutical products by generating a topologically diverse polymer library that competed with or outperformed currently available agents including generally regarded as safe (GRAS) excipients in terms of stabilization and cryoprotection abilities. The present investigation prioritized freeze-thaw cycles over long-term studies because it was focused on the stabilization throughout these structurally more severe events. Additionally, an aim was to have a well-defined baseline for each experiment, which was only possible with using the freshly prepared RNA-LNPs as internal reference. However, robust data on long term preservation may be useful for optimizing future endeavors.

[0206] For now, in the design of HPOD polymers, several factors were considered. There are no established design criteria reported for the development of macromolecular stabilizing agents. As a general principle, most of them have one of the key features, such as high polarity, charge, hydrophilicity, being zwitterionic, or distinct hydrophobic pockets in their molecular architecture (Murray & Gibson, 2022). In the context of cryopreservation, it was found beneficial to have a sheath of non-frozen bound water present in the molecule (Chapanian et al., 2012; Czechura et al., 2008). Thus, a new polymer which has large number of hydroxyl groups in a branched poly ether backbone was synthesized. The low intrinsic viscosity, compact nature and high biocompatibility shown by HPOD is due to its branched structure and is consistent with reports of branched polymer structures offering notable advantages over linearstructures (Abbina et al., 2017). Additional criteria taken into account were the high water solubility and the potential ability to bind unfreezable water molecules. It is believed that the HPODs have sugar-like hydration (see, for example: the MALDI data) and behavior at lower molecular weights; and function similar to other hydrophilic macromolecules at higher molecular weights used for cryopreservation. While not wishing to be limited by theory, this is most likely the reason that it provides good preservation for a wide variety of biotherapeutics at a range of physical stressors. In the case of heat protection, it was hypothesized that the polymers form a hydration shell around the biomolecules, which could help to maintain their native structure by stabilizing hydrogen bonds and preventing denaturation due to heat. Additionally, the increased viscosity compared to aqueous buffer reduces the molecular motion and thus decreases heat stress. The highly hydrated polyether structure of HPOD, is shown to imparts this property. Additionally, the polyol structure of HPOD serves the purpose of mimicking sugar molecules, which are known to have preservation properties (Kaushik & Bhat, 2003). The utilization of a sugar-mimicking polymers such as HPOD over sugars provides notable advantages, such as lower osmolarity and enhanced stability while allowing for fine tuning of the preservation capability, particularly when higher concentrations of cryoprotectant are needed. Additional long-term studies may be useful to fully realize the potential of this new excipient.

[0207] The present studies also identified a key feature, polymer topology, which has not previously been explored in the development and production of cryo- and lyo-protecting macromolecular excipients. The present findings demonstrated that the introduction of linear units within the branched structure had a remarkable effect on the amount of bound water molecules associated with HPOD (FIGs. 60-64) compared to similar polyether and polyol building blocks such as HPG and PGD (chemically identical but with a difference in topology). Further, a sharp increase was found in the amount of bound water molecules with decreasing degree of branching until about 42% (FIG. 61). The inventors believe that a low branching density and the resulting flexibility allowed for the formation of an optimal water structure within and around HPOD. The chain confinement presents in PGD (100% branching) resulted in a significant drop in the bound water (76% decrease; FIG. 62 and Table 7) possibly, while not wishing to be limited by theory, due to steric factors as the dendrimer interior cannot accommodate such large amount of water molecules even though the theoretical possibility of hydrogen bonding with water exists. A synergy of all these effects resulted in the exceptional hydration of HPOD, which was as high as 27 bound water molecules per monomer unit,higher than any known molecule. The inventors believe that structural features, such as linear units within branched structures which provide increased space for water molecule binding, chain confinement due to branching, and the polyether-polyol structure that facilitates extensive hydrogen bonding are all contributing towards the generation of a stable non- freezing bound water layer around and within the HPOD resulting in high hydration. The hydrated molecular ions seen in MALDI-ToF spectra of HPOD are further demonstrating that the water molecules are very tightly bound to HPOD. Such water adducts are rarely seen with polymer ions, they are more common for sugars (Kosyakov et al., 2019).

[0208] Based on the present findings, while HPOD exhibited similar performance to sucrose and HES, it did not match the efficacy of PVA in inhibiting ice recrystallization (FIGs. 63 and 64). Therefore, the inhibition of ice recrystallization alone does not provide a conclusive explanation for the performance of HPOD in preserving proteins and RNA-LNPs at very low temperatures. As ice recrystallization inhibition has been identified as an important process in the protection of sensitive material during a freeze-thaw cycle (Biggs et al., 2017; Mitchell et al., 2019; Deller et al., 2014), other mechanisms must be acting together to generate the preservation properties of HPOD. The generally accepted mechanisms with respect to macromolecular excipients in the context of protecting proteins and biotherapeutics from different external stresses are vitrification, preferential exclusion, and water replacement. All of these are associated with the alteration of the water structure around the proteins to preserve their native conformation and might be acting in the case of HPOD. With its extended hydration sphere, HPOD may form strong interactions with proteins and LNP components, providing a response to the external stimuli and thereby preserving the integrity of the stored cargo.

[0209] HPOD effectively solved many preservation challenges with a single polymer class. It also exhibits remarkable biocompatibility. The inventors anticipate that this would simplify the formulation process, reduce complexity, and potentially improve the stability and functionality of future LNP- and protein-based therapeutics for clinical applications. Although HPOD has remarkable properties, further immunogenicity studies, long term preservation studies, up-scaling of the synthesis and possibly improvement in the synthesis process may be useful to further validate these new macromolecular excipients for future clinical use.Example 2. Cryopreservation of mammalian cells in the presence of ultra-hydrating HPOD.I. Experimental

[0210] Cryopreservation of red blood cells (RBCs): Blood was collected from donors in ACD vacutainer tubes (BD). Blood was centrifuged at lOOOxg for 5 min and the supernatant was removed. The packed RBCs (80% hematocrit) were mixed with cryopreservation agents (250 pL) in a ratio of 1: 1 (V / V). Cry opreservation of RBCs was performed with HPOD-2 and 4 (175 mg / mL in Milli-Q water, 6.5 mg / mL of NaCl (final concentration)) and was compared with other stabilizing agents HES and glycerol that were made into 175 mg / mL solutions in water, including 3% mannitol and 6.5 mg / mL of NaCl (final concentrations). A cryotube (Coming, 1.2 mL) was loaded with cry opreservation agent (250 pL) and mixed with packed RBCs (250 pL) on a rocker for 30 min and placed under liquid nitrogen vapor for one week. After removing the samples from liquid nitrogen, all the samples were placed in water bath (37 °C) for 3 min. The samples were mixed gently and 5 pL of suspension from each sample was transferred into 295 pL Drabkin’s reagent. In another tube, the original cryopreserved samples after gently mixing were centrifuged for 5 mins at 3500 xg and 5 pL of the supernatant was transferred into 250 pL Drabkin’s reagent (triplicates). The absorbance of hemoglobilin (Hb) at 540 nm was measured on a plate reader for both RBC suspension and supernatant.

[0211] Hb concentration was calculated using the formula:Hemoglobilin concentration in g% Hb = OD540 x (final volume / sample volume) / 6.8 Percent of lysis = (Hb concentration in supematant / Hb concentration in suspension) x 100

[0212] Data was collected from three independent experiments (N = 3), performed in triplicates and is reported in average ± s.d.II. Results

[0213] The preservation of donated cells, tissues, and organs often demand much more sophisticated and viable approaches to improve the transplantation outcome. Notably, improved protocols for the cryopreservation of cells may, for example, eliminate many current logistical issues and increase the availability of cells for transplantation. Due to the ultra-hydrating nature, low molecular weight, low intrinsic viscosity, and excellent biocompatibility, the inventors hypothesized that HPODs might have a better cryoprotecting ability than cryoprotectants in current use. Thus, as a proof-of-concept, red blood cells (RBCs) were chosen to investigate the cry opreservation ability of HPODs. The viability ofcryopreserved RBCs in liquid nitrogen vapor was measured as the percentage of hemolysis in the presence of various cryoprotectants (FIG. 67 and FIG. 68). The performance of HPOD- 2 & 4 was compared to FDA approved cry opreservation agents (cell-permeable glycerol, noncell permeable hydroxy ethyl starch (HES), and polyvinyl alcohol (PVA)) in combination with mannitol using standard protocols for 24 h. HPOD-4 showed superior performance than HPOD-2 and equally effective compared to glycerol, HES, and PVA without using mannitol (FIG. 68). Further, cry opreservation of RBCs for 7 days showed that HPOD-4 performed comparable to clinically used cry opreserving agents with additional advantages of being low molecular weight and low viscosity. Accordingly, HPODs may also be useful as cryoprotectants for cells, tissues and / or organs in addition to active agents.Example 3: Hyperbranched Lipo-polymer as Linear PEG Alternative in LNPs

[0214] An aim is to replace linear PEG-lipids with a polymeric hyperbranched lipid in RNA-LNPs. Due to their high hydration and low-immunogenic profde of the polymer the inventors hypothesize that this will not only avoid linear PEG-associated limitations but also enhance LNP stability during storage and handling. This strategy may, for example, offer a dual advantage of improving both immune-compatibility and formulation robustness for nextgeneration RNA therapeutics. Another aim is the encapsulation of drugs to produce long- circulating drug-loaded formulations. Two exemplary lipidic initiators were used (Scheme 3).Scheme 3. Exemplary lipidic initiators.

[0215] The lipidic initiators were used to polymerize OPD monomer and glycidol (control) in line with the procedures described in Example 1 to prepare HPOD polymer and HPG polymer, respectively. The molecular weights are shown in Table 8.Table 8

[0216] LNP preparation with HPOD lipo-polymers: Self-amplifying RNA lipid nanoparticles (saRNA-LNPs) encoding the luciferase protein were formulated by rapidly mixing an ethanol solution of lipids comprising PEG-lipid or lipopolymer (C16HPOD) with an aqueous buffer (pH 4.5) containing the saRNA. The N / P ratio was kept at 10. The resulting mixture was immediately diluted into phosphate-buffered saline (PBS) and subsequently purified using Ami con™ centrifugal filters with a 10 kDa molecular weight cut-off (MWCO). The final saRNA- LNP formulations were typically at concentrations ranging from 40 to 50 pg mL '.

[0217] Composition of lipids in LNP :1. SM-102 / DGPE / Chol / DMG-PEG-2000 (45: 17.5:36.25: 1.25).2. SM- 102 / DOPE / Chol / C 16HPOD (45 : 17.5 : 36.25 : 1.25).3. MC3 / DSPC / Chol / DMG-PEG-2000 (50:10:38.5:1.5).4. MC3 / DSPC / Chol / C16HPOD (50:10:38.5:1.5).

[0218] Liposome Preparation: Liposomes were prepared using the thin film hydration method. A lipid solution in ethanol containing PEG-lipid or lipopolymer was added to a glass vial, and a thin lipid film was formed by evaporating the solvent under a steady stream of nitrogen gas. Phosphate-buffered saline (PBS) was then added to hydrate the film, and the mixture was stored at 4 °C overnight. The following day, the dispersion was heated to 65 °C and subjected to three freeze-thaw cycles, resulting in the formation of liposomes.

[0219] Composition of lipids in Liposome:1. DSPC / Chol / DMG-PEG-2000 (65:30:5).2. DSPC / Chol / C16HPOD (65:30:5).3. DGPC / Chol / DMG-PEG-2000 (65:30:5).4. DOPC / Chol / C16HPOD (65:30:5).

[0220] Hydrodynamic meter (size) and zeta potential: The particle size and zeta potential of the prepared LNP / liposomes were measured at room temperature using a Zetasizer Nano ZS instrument (Malvern Instruments, UK).

[0221] Encapsulation Efficiency: Encapsulation efficiency was determined using the Quant-iT™ RiboGreen™ RNA Assay Kit (Thermo Fisher Scientific), following themanufacturer’s protocol. Encapsulation efficiency (%) was calculated as: (Total RNA - Free RNA) / Total RNA x 100.

[0222] Detection of lipo-polymer in LNP: Lipid separation was performed using a reverse-phase column on a liquid chromatography mass spectrometry (LC-MS) system. saRNA-LNP samples were initially dissolved in water and subsequently diluted with methanol. The resulting aliquots were directly injected into the LC-MS system for lipid profiling.

[0223] Results of the characterization are shown in FIGs. 69-78.

[0224] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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Claims

CLAIMS:

1. A hyperbranched poly ether polyol, comprising a combination of units selected from:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

2. The hyperbranched poly ether polyol of claim 1 , comprising the:(i) dendritic units (D) of the formula:(ii) semi-dendritic units (sD13-sD18) of the formula:(iii) semi-dendritic units (sD13-sD17) of the formula:(iv) linear units (LI 7) of the formula:(v) linear units (LI 3) of the formula:(vi) linear units (LI 8) of the formula:(vii) terminal units (T) of the formula:

3. The hyperbranched poly ether polyol of claim 1 or 2, further comprising a core of the formula:whereinX1, X2and X3are each independently Ci-4alkylene; andR1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

4. The hyperbranched polyether polyol of claim 3, wherein X1, X2and X3are all -CH2-.

5. The hyperbranched poly ether polyol of claim 3 or 4, wherein R1is ethyl.

6. The hyperbranched polyether polyol of claim 3 or 4, wherein R1is the lipidic group.

7. The hyperbranched polyether polyol of claim 5, wherein the lipidic group is of the formula:wherein R1' is C4-3oalkyl.

8. A method of preparing a hyperbranched polyether polyol, the method comprising polymerization of a compound of the structure:

9. The method of claim 8, wherein the method comprises anionic ring opening multibranching polymerization (ROMBP).

10. The method of claim 7 or 8, wherein the ROMBP comprises an initiator of the structure:whereinX1, X2and X3are each independently Ci-4alkylene; and R1is Ci-4alkyl or a lipidic group comprising at least 5 carbon atoms.

11. The method of claim 10, wherein X1, X2and X3are all -CH2-.

12. The method of claim 10 or 11, wherein R1is ethyl.

13. The method of claim 10 or 11, wherein R1is the lipidic group.

14. The method of claim 13, wherein the lipidic group is of the formula:wherein R1' is C4-3oalkyl.

15. A hyperbranched poly ether polyol prepared by a method as defined in any one of claims 8 to 12.

16. A hyperbranched polyether polyol prepared by a method as defined in claim 13 or 14.

17. The hyperbranched poly ether polyol of any one of claims 1 to 7, 15 or 16, having a degree of branching of from about 1% to about 99%.

18. The hyperbranched poly ether polyol of any one of claims 1 to 7, 15 or 16, having a degree of branching of from about 23% to about 46%.

19. A hyperbranched poly ether polyol having a degree of branching of from about 23% to about 46%.

20. The hyperbranched poly ether polyol of any one of claims 1 to 7 or 15 to 19, wherein the degree of branching is about 23%, about 29%, about 37%, about 42% or about 46%.

21. The hyperbranched poly ether polyol of any one of claims 1 to 7 or 15 to 20, having a number average molecular weight (Mn) of from about 640 to about 11,500 Da.

22. The hyperbranched poly ether polyol of any one of claims 1 to 7 or 15 to 21 having an intrinsic viscosity of from about 4 to about 15 mL / g.

23. A hyperbranched poly ether polyol having an intrinsic viscosity of from about 4 to about 15 mL / g.

24. A conjugate comprising a hyperbranched polyether polyol as defined in any one of claims 1 to 7 or 15 to 23 conjugated to a lipid.

25. The conjugate of claim 24, wherein the lipid comprises a phospholipid.

26. A lipid nanoparticle comprising (1) a hyperbranched poly ether polyol as defined in any one of claims 6, 7 or 16, or any one of claims 17, 18, and 20 to 22, wherein in the hyperbranched poly ether polyol, R1is as defined in any one of claims 6, 7 or 16; or (2) a conjugate as defined in claim 24 or 25.

27. The lipid nanoparticle of claim 26, wherein the lipid nanoparticle further comprises an ionizable lipid, a phospholipid and cholesterol.

28. The lipid nanoparticle of claim 26 or 27, further comprising an active ingredient.

29. A liposome comprising (1) a hyperbranched polyether polyol as defined in any one of claims 6, 7 or 16, or any one of claims 17, 18, and 20 to 22, wherein in the hyperbranched poly ether polyol, R1is as defined in any one of claims 6, 7 or 16; or (2) a conjugate as defined in claim 24 or 25.

30. The liposome of claim 29, wherein the liposome further comprises a phospholipid and cholesterol.

31. The liposome of claim 29 or 30, further comprising an active ingredient.

32. A composition comprising a hyperbranched polyether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25.

33. The composition of claim 32, further comprising an active ingredient.

34. A pharmaceutical composition comprising (1) an active ingredient and a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or the conjugate of claim 24 or 25; (2) a lipid nanoparticle as defined in claim 28; or (3) a liposome as defined in claim 31.

35. The pharmaceutical composition of claim 34, wherein the pharmaceutical composition comprises the hyperbranched polyether polyol as defined in any one of claims 1 to 7 or 15 to 23, the pharmaceutical composition further comprises a lipid nanoparticle and the active ingredient is encapsulated in or conjugated to the lipid nanoparticle.

36. The pharmaceutical composition of claim 35, wherein the lipid nanoparticle comprises an ionizable lipid, a phospholipid, cholesterol and a linear PEG-lipid.

37. The pharmaceutical composition of claim 34, wherein the pharmaceutical composition comprises the hyperbranched poly ether polyol or the conjugate as defined in claim 24 or 25, and wherein the hyperbranched poly ether polyol is as defined in any one of claims 6, 7 or 16, or any one of claims 17, 18, and 20 to 22, wherein in the hyperbranched poly ether polyol, R1is as defined in any one of claims 6, 7 or 16.

38. The lipid nanoparticle of claim 28, liposome of claim 31, composition of claim 33 or pharmaceutical composition of any one of claims 34 to 37, wherein the active ingredient comprises a drug.

39. The lipid nanoparticle of claim 28, liposome of claim 31, composition of claim 33 or pharmaceutical composition of any one of claims 34 to 37, wherein the active ingredient comprises a nucleic acid or an antisense oligonucleotide (ASO).

40. The lipid nanoparticle of claim 28, liposome of claim 31, composition of claim 33 or pharmaceutical composition of any one of claims 34 to 37, wherein the active ingredient comprises a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA).

41. The lipid nanoparticle or pharmaceutical composition of claim 40, wherein the ribonucleic acid comprises transfer RNA (tRNA), messenger RNA (mRNA), selfamplifying RNA (saRNA) or small interfering RNA (siRNA).

42. The lipid nanoparticle of claim 28, liposome of claim 31, composition of claim 33 or pharmaceutical composition of any one of claims 34 to 37, wherein the active ingredient comprises a protein or peptide.

43. Use of a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or conjugate as defined in claim 24 or 25 as a cryoprotectant, lyoprotectant or stabilizer or preservation agent or a hydrating agent or as an excipient.

44. The use of claim 43, wherein the hyperbranched polyether polyol or conjugate is for use in combination with an additional cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient.

45. The use of claim 43, wherein the hyperbranched polyether polyol or conjugate is for use as the sole cryoprotectant, lyoprotectant or stabilizer or preservation agent or hydrating agent or excipient.

46. The use of any one of claims 43 to 45, wherein the cryoprotection or lyoprotection is of an active ingredient as defined in any one of claims 38 to 42.

47. The use of any one of claims 43 to 45, wherein the cryoprotection is of an organelle, a multicellular assembly, an organoid, a multicellular specimen, a cell, a tissue or an organ.

48. The use of any one of claims 43 to 45, wherein the stabilization is of a peptide or protein.

49. The use of claim 48, wherein the peptide or protein is a biotherapeutic.

50. Use of a lipid nanoparticle as defined in claim 28, liposome as defined in claim 31, or a pharmaceutical composition comprising a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25 and an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject.

51. Use of a lipid nanoparticle as defined in claim 28, liposome as defined in claim 31, or a pharmaceutical composition comprising a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25 and an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable by the active ingredient in a subject.

52. The use of claim 50 or 51, wherein the lipid nanoparticle, liposome or pharmaceutical composition is cryopreserved then thawed prior to use in the subject.

53. The use of claim 50 or 51, wherein the lipid nanoparticle, liposome or pharmaceutical composition is lyophilized and reconstituted prior to use in the subject.

54. A use of an active ingredient for treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by a composition as defined in claim 33.

55. A use of an active ingredient in the preparation of a medicament for treatment of a disease, disorder or condition treatable by the active ingredient in a subject, wherein prior to use in the subject, the active ingredient has been cryoprotected, lyoprotected or stabilized by a composition as defined in claim 33.

56. The use of claim 54 or 55, wherein the composition is a pharmaceutical composition, and the hyperbranched poly ether polyol or conjugate is not removed prior to use in the subject.

57. The use of any one of claims 50 to 56, wherein the active ingredient is as defined in any one of claims 38 to 42.

58. The use of any one of claims 50 to 56, wherein the use is of a pharmaceutical composition as defined in claim 37, and wherein the active ingredient is encapsulated in or conjugated to a lipid nanoparticle comprising the hyperbranched polyether polyol or conjugate as defined in claim 26 or 27.

59. The use of any one of claims 50 to 58, wherein the subject is a human.

60. Use of a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25 as a food additive.

61. Use of a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 23 or 24 as a cosmetic additive.

62. A food comprising a hyperbranched polyether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25 as a food additive.

63. A cosmetic comprising a hyperbranched poly ether polyol as defined in any one of claims 1 to 7 or 15 to 23 or a conjugate as defined in claim 24 or 25 as a cosmetic additive.

64. The compound 3-(oxiran-2-ylmethoxy)propane-l,2-diol) (OPD).

65. Use of the compound of claim 59 in the preparation of a hyperbranched poly ether polyol.

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