Polysaccharides and oligosaccharides encapsulated in lipid nanoparticles

Abstract

The invention relates to a process for the preparation of a composition comprising an oligosaccharide or a polysaccharide encapsulated in lipid nanoparticles, the process comprising the following steps: preparing a first solution of a lipid portion comprising a phospholipid, a neutral lipid, and a lipid sterol; preparing a second solution comprising a poly- or oligosaccharide; preparing a third solution containing a buffer; mixing the first and second solutions using a microfluidic system to obtain lipid nanoparticles encapsulating the oligosaccharide or the polysaccharide; combining the third solution with the first and second solutions. The invention also relates to a composition comprising lipid nanoparticles obtainable by the above-defined process, said composition comprising a lipid portion comprising a phospholipid, a neutral lipid compound in which a lipid chain is bound to a glycol group, and a lipid sterol; a buffer; and an oligosaccharide or polysaccharide.

Description

Polysaccharides and Oligosaccharides Encapsulated in Lipid NanoparticlesField of the invention

[0001] The present invention relates to lipid nanoparticles comprising a polysaccharide and / or an oligosaccharide.Background of the Invention

[0002] In recent decades, so-called lipid-based drug delivery systems (LBDD) have attracted significant attention from researchers. These systems offer various advantages over conventional dosage forms, such as greater stability and bioavailability. Until recently, the most common of these systems was the liposome-based system. Liposomes consist of one or more lipid bilayers and a hydrophilic core. They can be used to deliver both hydrophobic substances, which localize within the lipid bilayer, and hydrophilic substances, which reside in the hydrophilic core. The size of liposomes generally ranges from a minimum of 50 nm to a maximum of approximately 1000 nm, and they are also suitable for encapsulating relatively large molecules.

[0003] Glycosaminoglycans are natural products of considerable pharmaceutical interest. Among the most widely used are heparin, dermatan, heparan sulfate, chondroitins, and hyaluronic acid. The molecular weight of these natural products varies significantly, generally ranging from 5 to 40 kDa for heparin, dermatan, heparan sulfate, and chondroitins, while it can reach up to 4,000 kDa in the case of hyaluronic acid. The high molecular weight of these compounds often makes oral administration impractical, necessitating intravenous administration. However, it is well known that intravenous administration leads to rapid degradation of the molecule, with a consequent rapid reduction in biological activity.

[0004] T.D. Kim et al. (Studies on liposome encapsulated heparin, Thrombosis Research 43; 603-612, 1986) disclose the formation of liposomes containing heparin. Although the article demonstrates an improvement in the stability of heparin once encapsulated in liposomes, the results are poorly reproducible, making the system unsuitable for pharmaceutical application.

[0005] More recently, another LBDD system has been developed, namely lipid nanoparticles (LNPs). These nanoparticles consist of a lipid outer shell containing reverse micelles that encapsulate the active ingredient. These systems have been designed for RNA fragments, DNA fragments, or active ingredients of limited size. Lipid nanoparticles are typically composed of four main lipids: an ionizable lipid in either a positively charged or neutral form, a saturated phospholipid (zwitterionic lipid) forming the bilayer, cholesterol, and a PEG-conjugated lipid.In Patisiran™, the molar ratio among the listed components (DLin-MC3-DMA: 1,2-DSPC: Cholesterol: C-DMC-PEG(2000)) is 50: 10:38.5: 1.5.

[0006] WO 2022 / 115604 discloses novel PEGylated lipid compounds to be used in compositions comprising, in addition to the PEGylated lipid, cholesterol and phospholipids for the encapsulation of mRNA fragments. The patent application specifies that the encapsulation may be used for RNA fragments or small molecules.

[0007] During the COVID-19 pandemic, the development of new vaccines led to the emergence of new high-shear microfluidic systems designed to produce LNPs with suitable size and poly dispersity index (PDI), capable of optimally encapsulating various nucleic acid cargos with high reproducibility and efficiency. These microfluidic systems include Impingement Jet Mixing (IJM) systems, Herringbone mixers, and high-shear microfluidic systems. The pandemic demonstrated that these systems constitute a vertically integrated LNP production platform enabling seamless transfer between alternative mixing technologies and a unified technology usable at all stages of the scalability process. Nevertheless, even in this case, the system is considered suitable only for DNA and RNA fragments or small molecules.

[0008] However, it is worth noting that vaccines are typically administered intramuscularly, whereas certain polysaccharides — such as glycosaminoglycans, and in particular heparin — are administered intravenously. All pharmacokinetic data obtained for one route of administration cannot be directly translated to a different administration route.

[0009] Recently, there has been increasing interest in, and approval of, drugs formulated using the LNP strategy, due to several advantages, including the delivery of both hydrophobic and hydrophilic drugs, controlled release, reduced drug-related side effects, biocompatibility, and increased drug half-life. However, these applications still concern only low-molecular-weight molecules.

[0010] WO2023084217A1 describes advanced payload delivery systems based on lipid nanoparticles functionalised with polymers (PF-LNPs), designed for intracellular delivery of nucleic acids, particularly self-amplifying RNA (saRNA). A distinguishing feature of the system described in the patent is the use of a preparation method different from standard microfluidic approaches, which adversely affects particle size distribution. Experimental data (e.g., Figure 3B, page 95) show that nanoparticles obtained via the proposed method exhibit PDI values higher than those typically achieved using microfluidic methods (typically < 0.2).

[0011] There is therefore a need for new methods for the encapsulation of poly- and oligosaccharides that enhance their stability for intravenous administration.Summary of the Invention

[0012] It has been surprisingly found that it is possible to prepare lipid nanoparticles comprising a polysaccharide or an oligosaccharide having a molecular weight ranging from 1,200 Da to 200,000 Da by mixing, using a microfluidic system, a solution comprising a neutral lipid compound, a neutral phospholipid, a sterol, and optionally an ionizable cationic lipid compound, with a solution comprising the oligosaccharide or the polysaccharide. Preferably, the polysaccharides and oligosaccharides incorporated in the lipid nanoparticles have a molecular weight ranging from 1,500 to 150,000 Da, more preferably from 2,000 to 120,000 Da.

[0013] The invention also relates to lipid nanoparticles comprising an oligosaccharide or a polysaccharide obtainable by the process defined above.Detailed Description of the Invention

[0014] The neutral lipid compounds usable in the present invention are compounds containing at least one lipid chain bound to a polyglycol group. When polyethylene glycol is used as the polyglycol, the compounds are referred to as PEGylated lipids. The bond between the lipid group and the glycol group may be formed by esterification between the carboxyl group of the lipid and one or two hydroxyl groups of the terminal glycol group, as in the case of DMG- PEG(2000), or may involve a functional group linking the polyglycolic part to the lipid part, as in DSPE-MPEG(2000). In a preferred embodiment, DMG-PEG(2000) is used. One of the purposes of the neutral lipid compounds is to prevent the adsorption of plasma proteins, thereby inhibiting uptake by the mononuclear phagocyte system, which is one of the main barriers to the efficacy of LBDD systems.

[0015] Lipidic sterols are generally used in LNP systems to fill membrane packing defects. Among the sterols usable in the present invention, the most common is cholesterol, although similar molecules such as cholesterol sulfate may also be used, as well as cholesterol derivatives modified chemically to be pH-sensitive and / or capable of reacting with proteins and / or enzymes.

[0016] A wide variety of phospholipids may be used. The phospholipid is preferably selected from 1,2-distearoylphosphatidylcholine (1,2-DSPC), phosphatidylcholine, and phosphatidylethanolamine. The most commonly used phospholipid is 1,2-DSPC, which is a natural constituent of cellular membranes.

[0017] An optional component in the preparation of the LNPs is an ionizable cationic lipid. These compounds are neutral at approximately pH 7 and acquire a positive charge at pH values below their pKa, facilitating the formation of reverse micelles that encapsulate theglycosaminoglycan in the LNP core. Their neutrality at pH 7 results in much lower cytotoxicity compared to permanently charged cationic lipids. Preferred ionizable cationic lipids include: l,2-dioleoyl-3 -dimethylammonium propane (DODAP), l,2-dioleyl-3 -dimethylammonium propane (DODMA), 4-(dimethylamino)-butanoic acid-l-(9Z,12Z)-9,12-octadecadien-l-yl- 10, 13 -nonadecadi en-l-yl ester (DLin-MC3-DMA), 6-((2-hexyldecanoyl)oxy)-N-(6-((2- hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium (ALC-0315), and 306OH0 having formula

[0018] The preferred ionizable cationic lipids are DLin-MC3-DMA and 306OH0, with DLin- MC3-DMA being most preferred.

[0019] The buffer used in the present invention may be selected from a wide range of buffers. Non-limiting examples of suitable buffers include phosphate-buffered saline (PBS) and citrate buffer.

[0020] In order to obtain nanoparticles with stable and reproducible size, it is important to use suitable micro-mixing systems. Various systems have been developed to achieve this result, such as Impingement Jet Mixer (IJM), in which the flows of the two solutions — containing the lipid substances and the compound to be encapsulated — collide at high velocity and at a defined angle, as well as Herringbone mixers and high-shear microfluidic systems.Microfluidic systems usable in the present invention are disclosed by Masatoshi Maeki et al., Microfluidic technologies and devices for lipid nanoparticle-based RNA delivery, Journal of Controlled Release 344 (2022) 80-96, incorporated herein by reference.

[0021] The nanoparticles of the present invention can efficiently encapsulate oligosaccharides and polysaccharides. Non-limiting examples of encapsulatable polysaccharides include glycosaminoglycans (hyaluronic acid, heparin, dermatan, dermatan sulfate, chondroitin 4 sulfate, chondroitin 6 sulfate, keratan sulfate, heparan sulfate, glycosplit heparin), inulin, pectin, alginate, dextrans, sulfated dextrans, chitosan, chitin, arabinoxylans, arabinogalactans, carrageenan, pentosan polysulfate, glucomannans, and galactomannans. These polysaccharides may be chemically modified by the introduction of functional groups and / or conjugated with biologically active molecules.

[0022] The nanoparticles according to the invention are obtained through the following process. Three solutions are prepared: a first solution containing the lipid portion, i.e., a phospholipid, a neutral lipid, and a lipid sterol, and optionally an ionizable cationic lipid; a second solution containing a poly- or oligosaccharide; and a third solution containing a buffer.

[0023] The first two solutions are mixed together using a microfluidic system to obtain nanoparticles encapsulating the poly- or oligosaccharide. In a preferred embodiment, the microfluidic mixing system is an IJM (Impingement Jet Mixer).

[0024] The nanoparticles obtained using these microfluidic systems are characterized by very high stability in their physical properties, and therefore exhibit high reproducibility, allowing for the production of a consistent product over time. In particular, the volume-weighted average diameter of the nanoparticles according to the invention ranges from 40 nm to 200 nm, more preferably from 50 nm to 150 nm, and even more preferably from 60 nm to 120 nm.

[0025] Furthermore, the process according to the invention enables a high encapsulation efficiency of the poly- or oligosaccharide within the nanoparticles. Preferably, the encapsulation efficiency is greater than 50%, more preferably greater than 60%.

[0026] The composition obtained by the process of the present invention may be used as such, i.e., containing non-encapsulated poly- or oligosaccharide, or, for certain applications, purification of the composition may be recommended to remove the non-encapsulated poly- or oligosaccharide. Additionally, both the purified and non-purified compositions may be used in combination with other poly- or oligosaccharides optionally modified by the introduction of functional groups that render them pH-sensitive and / or capable of interacting with proteins and / or enzymes.Experimental Part

[0027] Lipid nanoparticles were prepared using a Nano Scaler IJM device from Knauer as follows.Pump 1 : solution of a polysaccharide in continuous flow.Pump 2: lipid solution loaded into a 1 mL loop.Pump 3 : buffer solution, in continuous flow.

[0028] The polysaccharide solution was prepared by dissolving the polysaccharide in a 0.14 mM or 0.014 mM NaCl solution, or in a 0.01 mM or 0.1 mM citrate buffer, and maintaining the solution under stirring overnight.Table 1

[0029] The solution containing the lipid portion was obtained by dissolving the lipid compounds in absolute ethanol at the concentrations reported in Table 1.

[0030] The following buffer solutions were prepared:• 150 mM phosphate-buffered saline (PBS), pH 7.4: approximately 8 g of sodium chloride, 0.2 g of potassium chloride, approximately 1.44 g of disodium phosphate, andapproximately 0.245 g of monopotassium phosphate were added to 1 L of deionized water. The pH was adjusted to the desired final value using HC1 or NaOH.• 15 mM phosphate-buffered saline (PBS), pH 7.4: prepared by tenfold dilution of the 150 mM solution.• 140 mM sodium chloride: approximately 8.18 g of sodium chloride were added to 1 L of deionized water. The pH was adjusted using HC1 or NaOH.• 14 mM sodium chloride: approximately 0.818 g of sodium chloride were added to 1 L of deionized water. The pH was adjusted using HC1 or NaOH.• 25 mM sodium acetate buffer, pH 5.5: approximately 1.839 g of sodium acetate and approximately 155.2 mg of acetic acid were added to 1 L of deionized water. The pH was adjusted using HC1 or NaOH.• 25 mM citrate buffer, pH 5.5: approximately 5.171 g of disodium citrate dihydrate and approximately 1.425 g of citric acid were added to 1 L of deionized water. The pH was adjusted using HC1 or NaOH.• 50 mM citrate buffer, pH 5.5: approximately 10.342 g of disodium citrate dihydrate and approximately 2.85 g of citric acid were added to 1 L of deionized water. The pH was adjusted using HC1 or NaOH.Examples of Lipid Nanoparticles with Unfractionated Heparin (UFH)

[0031] A suitable amount of UFH was dissolved in 0.14 mM NaCl for 2 hours at room temperature to reach the final concentration indicated in the table. PBS at pH 7.4 was used as the quench solution. The lipid mixture was prepared by adding 16 pL of a stock solution of DLin-MC3-DMA (100 mg / mL), 16 pL of a stock solution of cholesterol (5 mg / mL), 16 pL of a stock solution of 1,2-DSPC (25 mg / mL), and 189 pL of a stock solution of 1,2-DMG- PEG (1 mg / mL). The mixture was brought to a final volume of 1 mL with ethanol. LNPs were prepared using an IJM NanoScaler under condition 1. The total flow rate (TFR) was set at 3 mL / min with the following settings: Pump 1 (UFH): 1.5 mL / min, Pump 2 (Lipids): 0.5 mL / min, Pump 3 (Quench): 1 mL / min. Particle size and Zeta potential (Zp) were determined by dynamic light scattering (DLS) measurements on the formulations. A volume of 80 pL of solution was used for particle size analysis, while 800 pL were used for Zp determination. Encapsulation efficiency was assessed by HP-SEC-TDA, after measuring the UFH concentration outside the LNPs (recovered concentration). The encapsulation efficiency (EE%) was then calculated using the following formula:Table 2PDI= poly dispersity index; PBS = phosphate-buffered saline; Zp = zeta potential Flow rate ratios between pumps: 1.5 (Pump 1) : 0.5 (Pump 2) : 1 (Pump 3) **UFH was dissolved in PBS at pH 7.4

[0032] Table 2 reports the data from experiments conducted with UFH under various conditions and at different UFH concentrations, using lipid mixtures A to H as described in Table 1.

[0033] One millilitre of the formulation was centrifuged using a HERMLE Z326K centrifuge and a 100 kDa centrifugal filter, and washed with 8 mL of water. A 300 pL aliquot of the filtrate was directly injected into an HPLC system equipped with a UV / Vis detector. UV analysis of the ultrafiltered samples and HPLC of the formulation confirmed the results obtained by HP-SEC-TDA.

[0034] Additional experiments were conducted using low molecular weight heparin (LMWH). A suitable amount of LMWH was dissolved in 0.14 mM NaCl for 2 hours at room temperature to reach the final concentration reported in Table 3. Table 3 summarizes the obtained results.Table 3

[0035] Experiments were conducted using an ultra-low molecular weight heparin, commercially known as Arixtra™. A suitable amount of Arixtra™ was dissolved in 0.14 mM NaCl for 2 hours at room temperature to reach the final concentration reported in Table 4.Table 4

[0036] An experiment was conducted using hyaluronic acid (HA). An appropriate amount of HA was dissolved in PBS at pH 7.4 and left under stirring overnight. Table 5 presents the results obtained.

[0037] Experiments were also conducted using pentosan polysulfate (PPS). A suitable amount of PPS was dissolved in 0.14 mM NaCl for 2 hours at room temperature to reach the final concentration reported in Table 6. Table 6 summarizes the results obtained.Table 5Table 6

Claims

Claims1. A process for the preparation of a composition comprising an oligosaccharide or a polysaccharide encapsulated in lipid nanoparticles, the process comprising the following steps: a. preparing a first solution of a lipid portion comprising a phospholipid, a neutral lipid, and a lipid sterol; b. preparing a second solution comprising a poly- or oligosaccharide; c. preparing a third solution containing a buffer; d. mixing the first and second solutions using a microfluidic system to obtain lipid nanoparticles encapsulating the oligosaccharide or the polysaccharide; e. combining the third solution with the first and second solutions.

2. The process according to claim 1, wherein the microfluidic systems are selected from: an IJM (Impingement Jet Mixing) system, a Herringbone mixer, and a high-shear microfluidic system.

3. The process according to any one of claims 1-2, wherein step e) is carried out simultaneously with or after step d).

4. The process according to any one of claims 1-3, wherein the lipid portion further comprises an ionizable cationic lipid.

5. The process according to any one of claims 1-4, wherein the oligosaccharide or polysaccharide is selected from: a glycosaminoglycan, inulin, pectin, alginate, dextrans, sulfated dextrans, chitosan, chitin, arabinoxylans, arabinogalactans, carrageenan, pentosan polysulfate, glucomannans, galactomannans.

6. The process according to claim 5, wherein the glycosaminoglycan is selected from: hyaluronic acid, heparin, dermatan, dermatan sulfate, chondroitin 4 sulfate, chondroitin 6 sulfate, keratan sulfate, heparan sulfate, and glycosplit heparin.

7. The process according to any one of claims 1-6, wherein the phospholipid is selected from 1,2-distearoylphosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine, and the lipid sterol is cholesterol.

8. The process according to any one of claims 1-7, wherein the microfluidic system is an IJM system.

9. A composition comprising lipid nanoparticles obtainable by the process of any one of claims 1-8, said composition comprising: a. a lipid portion comprising a phospholipid, a neutral lipid compound in which a lipidchain is bound to a glycol group, and a lipid sterol; b. a buffer; and c. an oligosaccharide or polysaccharide.

10. The composition according to claim 9, wherein the lipid nanoparticles have a volume- weighted average diameter between 50 and 200 nm.

Images