EP4739283A1

EP4739283A1 - Peptide stereocomplexation with biodegradable stereoregular polyesters in water and hydrogel forms thereof

Info

Publication number: EP4739283A1
Application number: EP24742694.3A
Authority: EP
Inventors: Abraham Yaacov DOMB
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2023-07-05
Filing date: 2024-07-03
Publication date: 2026-05-13
Also published as: WO2025008819A1

Abstract

The application discloses a stereocomplex of a hydrophilic stereoregular biodegradable polyester and a peptide active agent or a protein for delivery of peptides or proteins in vivo.

Description

PEPTIDE STEREOCOMPLEXATION WITH BIODEGRADABLE STEREOREGULAR POLYESTERS IN WATER AND HYDROGEL FORMS THEREOF

FIELD OF INVENTION

The technology disclosed herein generally concerns stereocomplexes of peptides and proteins with water soluble stereoregular polymers for controlled peptide delivery. Of particular interest are water soluble block copolymers of D-PLA with water-soluble polymers, including polyethylene glycol (PEG) and polysaccharides and compositions thereof for use in therapeutic peptides delivery and hydrogel formation.

BACKGROUND

Over one hundred peptide drugs, including insulin, LHRH, somatostatin and TRH, have been clinically used for treating various diseases. Advances in the synthesis of recombinant peptides, proteins, DNA and RNA, as well as the exquisite specificity and reduced side effects inherent in their use have raised interest in their clinical development. Peptides present multiple benefits as potential drugs. These include high selectivity, high potency, low toxicity and low accumulation in tissue. However, due to large size, low stability, enzymatic degradation and poor permeability through biological membranes, getting appropriate extended release for days and weeks after subcutaneous or intramuscular injection, absorption and high bioavailability whether by oral or mucosal routes of administration is still a challenge.

Biodegradable polymers such as poly(lactic-glycolic acid) PLGA have been used for the extended delivery of leuprolide (Lupron Depot® and Zoladex), exenatide (Bydureon®), and octreotide (Sandostatin® LAR). Other strategies explored oral and nasal spray formulations (Miacalcin), inhalation devices (Afrezza), and transdermal patches but they did not lead to lasting commercial products. Beside the above mentioned few formulations for the delivery of peptides, most peptides are delivered by injection for immediate action and no other delivery systems for extended delivery, oral delivery or nasal and pulmonary delivery of peptide drugs are known.

Biodegradable protein-based gels have been reported for medical and other applications. They are usually made by chemical crosslinking gelatin and other types of natural proteins to form gels.

SUMMARY OF INVENTION

Peptide therapeutics have gained interest in recent years due to their high specificity and reduced side effects. However, poor stability and low bioavailability by an administration mode other than injection has been proven limited; consequently, limiting development of slow or sustained release modalities. To improve delivery of peptide drugs and similar active agents, two practical limitations had to be overcome. The first limitation was the limited control over the release profile of the drug entity; and the second its presentation as a stable drug entity that could sustain different administration modes and formulations.

The technology disclosed herein provides means for overcoming both limitations by providing solid forms of peptides which are stable, can be administered by a variety of administration modes and importantly enable quantified sustained or delayed release in vivo. The solid forms of the invention are stereocomplexes of two complementing materials, one being a peptide and the other a polymer that due to their opposite configurations interlock to form a stable composite, demonstrating improved physical and pharmaceutical properties. These solid forms may be caused to shed off the polymeric component or decompose to release the peptide in a controlled manner.

The composite materials of the invention may be characterized by presence of a properly selected polymer that contributes to the stability of the composite and which is also susceptible to hydrolysis in a controlled manner. The composite materials are further unique in having a surface charge or a surface polarity that is derived from surface-exposed hydrophilic functionalities making up the polymer and/or the peptide agent. The presence of the surface-exposed functionalities not only contributes to improving the solubilization of the peptide in a variety of formulations, but also protects the composite from early or spontaneous degradation.

In exemplified systems disclosed herein, water-soluble D-PLA-based copolymers, such as D-PLA-PEG di- and tri-block copolymers and D-PLA grafted dextran, were treated with insulin, LHRH, somatostatin and semaglutide, separately, to form particles (e.g., of a size between 100 and 400 nm) of the corresponding stereocomplexes composites. The process leading to the formation of the stereocomplexes was characterized by yield, DSC and turbidity. The affinity between the polymer, e.g., D-PLA-PEG and the peptide, e.g., insulin, was demonstrated by Micro Scale Thermophoresis (MST). In vitro release of insulin from the formed stereocomplex in PBS pH 7.4 solution, showed sustained release over a period of 80 days. Subcutaneous administration of the stereocomplex particles to diabetic mice exhibited reduced blood glucose levels over 80 days.

The use of a water-soluble polymer, such as D-PLA-PEG, for forming a stereocomplex in water expands the potential use of this method as a method inducing stabilization of peptides as well as their effective delivery via the oral, pulmonary and nasal modes of administration. Other water soluble stereoregular polymers may include poly(oc-hydroxy esters) derived from hydrophilic amino-acids. Copolymerization with D-lactic acid results in a water-soluble D-configured polyesters. Another class of water-soluble polyesters is based on tartaric acid where diethyl tartarate containing two hydroxyl groups polymerized with hydroxyl protected tartaric acid to form fully tartaric acid based stereoregular polyesters suitable for stereocomplexation with peptides.

Thus, in its broadest scope, the invention provides a stereocomplex of a stereoregular polymer and an active agent such as a peptide drug, wherein the polymer and the active agent having opposite configurations and wherein the stereocomplex having exposed hydrophilic functionalities.

In a first of its aspects the invention provides a stereocomplex of a stereoregular polymer and peptide active agent, the stereocomplex having outwardly extended hydrophilic functional groups.

The invention further provides a solid composite of a polymer and a peptide drug, wherein the composite is a hydrolysable stereocomplex of the polymer and the peptide drug, and wherein hydrophilic functionalities of the polymer and/or peptide are extended outwardly from the composite surface.

Also provided is a particulate material having surface-extending (or surface-exposed) plurality of hydrophilic functionalities, wherein the material is a stereocomplex of a stereoregular polymer and a peptide drug, and wherein the hydrophilic functionalities are functionalities present on the polymer and/or the peptide drug.

In some embodiments of materials of the invention, the stereocomplex is used for forming a gel for medical or non-medical uses.

The stereocomplex is typically a complex of two materials of opposite stereoconfigurations or of complementary configurations, wherein one of the materials is a stereoregular polymer and the other is an active agent, typically a peptide drug. The interaction between the materials, resulting in complexation, may be a stereoselective van der Waals interaction, wherein the polymer and the active agent interact through at least a region of either material to provide a stereoselective complex. The stereocomplexation need not involve the full structural backbone of either material. In fact, as depicted in Fig. 1, the peptide may interact with some of the peptide or protein chains, mainly with those exposed for interaction; thereby not affecting the 3D structure of the active agent. In other words, as long as the peptide has at least one region, segment or functionality of a stereo-configuration that is opposite to that of the polymer, a stereocomplex may be formed. Protein drugs having 3D structures that may deteriorate during complexations do not make part of the present invention.

As the interaction between the polymer and the active agent is stereoselective, namely occurring between two materials of opposite configurations, the interaction is by no means limited to the generation of 1: 1 complexes. The stereocomplexation may yield stable stereocomplexes between a peptide or protein and a biodegradable stereoregular polyesters, namely complexes in which the weight ratio of peptide drug (or inactive protein)-to-polymer may be from about 1: 1 to greater than 1:100 or 100:1, for example between 1: 1 and 5:1 (active agent: polymer). Thus, in some cases, a stereocomplex of the invention may comprise an active agent and a polymer at a molar ratio between 1 : 1 and 5: 1, or a weight ratio that is 1 :1, or 2:1, or 3: 1, or 4:1 or 5:l (active agent: polymer). The interaction between the peptide active agent or a non-active protein such as gelatin, and the polymer is reversible, permitting selective dissociation, degradation or otherwise stripping off of the polymer from the active agent. However, the dissociation may not be spontaneous; rather may be triggered and rate-controlled. The selective dissociation of the polymer from the active agent may be achievable by hydrolysis in an aqueous medium. For example, hydrolysis of a polyester polymer in water decreases the stereoregular chain length to a point where the polyester block is short enough to detach from the peptide. Chain length of a trimer of D-PLA is usually short enough to detach from the stereocomplex. The stereocomplex detachment can be affected by a solvent such as DMSO and DMF, change in ionic strength or change in acidity of the media, pH change and heat. In these cases, the detachment of the stereocomplex may take place without being degraded.

In a stereocomplex of the invention, the polymer and the peptide active agent are of opposite configurations or are of complementary configurations. In other words, each of the materials having a spatial arrangement that may be regarded complementary. As the two materials forming a stereocomplex according to the invention may differ substantially in size and spatial arrangement, their configurations may be considered relative configurations and may be differ in their D/L designations. One of the materials may be of the D configuration while the other may be of the L configuration, thus being of opposite configurations and accordingly complementary to each other.

In some embodiments, the polymer is a D-polymer. In other embodiments, the polymer is a L-polymer.

In some embodiments, the active agent is of the D configuration and the polymer is a L-polymer. In some embodiments, the active agent is of the L configuration and the polymer is a D-polymer.

The polymer used in stereocomplexes of the invention is a “stereoregular” polymer having a stereochemical regularity in the sequential repeating units. The polymer comprises an ordered arrangement of monomers and/or an ordered arrangement of pendent groups along the polymer backbone. Non-limiting examples of stereoregular polymers include stereoregular polylactic acid (PLA), PLA block copolymers with glycolic acid, racemic lactic acid, caprolactone, ethylene carbonate, polyethylene glycol, poly(2-hydroxy butyrate), and poly(2-hydroxy butyrate) block copolymers. Other suitable stereoregular polymers include polyesters of alpha hydroxy acids derived from amino acids and polymers based of tartaric acid.

The polymer may be provided as a homopolymer or conjugated or associated with one or more additional polymers or extension groups. In some embodiments, the polymer is a block copolymer of a homopolymer and a hydrophilic polymer, such as polyethylene glycol, polypropylene glycol and their copolymers, saccharides and polysaccharides.

Unlike the polymer, which may be stereoregular, the peptide active agent need not have a sequential configuration. The active agent is a peptide. The peptide is a drug entity or a cosmetically or agriculturally effective amino acid polymer having between 5 and about 50 amino acids. However, for the purpose of preparing peptide-based hydrogels, proteins such as gelatin and plant proteins may be used as the 3D structure.

In some embodiments, the peptide may be a unit comprised of polypeptides (one or more polypeptides) that are physically associated with one another and function together as a discrete functional unit, for example, insulin. In some cases, the peptide may comprise multiple polypeptides that are assembled to form a multimer, e.g., a homo-oligomer, or a hetero-oligomer. The peptide may be a naturally occurring, or a wild type peptide, or a modified, or a non-naturally occurring peptide. The peptide may have a structure different from a wild type peptide by the addition, substitution or deletion of one or more amino acids. The peptide may be in a folded conformation having a secondary or tertiary structure.

In some embodiments, the active agent is a peptide selected amongst drug peptides used in medicine. The peptide may thus be selected from antibiotic peptides, anticancer peptides, cardiovascular peptides, endocrine peptides, antiviral peptides, antibacterial peptides, antifungal peptides, gastrointestinal peptides, opiate peptides, plant peptides, respiratory peptides, vaccine peptides and others. Non-limiting examples may be selected amongst dipeptide, tripeptide, tetrapeptide, pentapeptide and longer peptides; as well as from oligopeptide, polypeptide, proteins, lipopeptide, neuropeptides, and others.

In some embodiments, the peptide is selected amongst peptide hormones such as adrenocorticotropic hormone (ACTH), endorphine, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormones, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin and vasoactive intestinal peptide (VIP).

In some embodiments, the active agent may be selected amongst insulin, somatostatin, semaglutide, exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, tirzepatide, growth hormones, growth factors, semaglutide, gelatin, collagen, desmopressin acetate, Ceruletide, Sincalide, Taltirelin, Protirelin, Sermorelin, Somatorelin, Tesamorelin, Secretin,, Thymalfasin, Thymopentin, Elcatonin, Human Calcitonin, Teriparatide, Atosiban, Carbetocin, Oxytocin, Buserelin, Gonadorelin, Goserelin, Histrelin, Leuprolide, Nafarelin, Triptorelin, Abarelix, Cetrorelix, Degarelix, Ganirelix, Depreotide, Edotreotide, Lanreotide, Octreotide, Pentetreotide, Vapreotide, Argipressin, Desmopressin, Lypressin, Phenypressin, Terlipressin, Enfuvirtide, Glatiramer, Ziconotide, Saralasin, Bivalirudin, Eptifibatide, Carperitide, Nesiritide, Icatibant, Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Pramlintide, Linasclotide, Sinaspultide, Pasireotide, Teduglutide, Peginesatide, Pentagastrin, Aviptadil and salt forms thereof.

In some embodiments, the stereocomplex comprises (i) a polymer selected from stereoregular D-polylactic acid (D-PLA), D-PLA block copolymers with glycolic acid, racemic lactic acid, caprolactone, ethylene carbonate, polyethylene glycol, poly(2-hydroxy butyrate), and poly(2-hydroxy butyrate) block copolymers; or D-stereoregular polyphydroxy esters) derived from amino acids or polymers made from tartaric acid should be considered; and (ii) a peptide active agent.

In some embodiments, the active agent is as disclosed and selected herein.

In some embodiments, the polymer is PLA or comprising PLA blocks.

In some embodiments, the polymer and the peptide active agent are chemically associated through an ionic or a covalent bond; and are further associated to form the stereocomplex. In some embodiments, the stereocomplex is D-PLA and the active agent is in the L configuration. The D-PLA may be of at least 7 lactic acid monomer units. In some embodiments, the D-PLA comprises between 7 and 20 lactic acid monomer units.

In some embodiments, the stereocomplex is of a D-PLA block copolymer with a polymer such as polyethylene glycol (PEG), polypropylene glycol (PPG) and their copolymers, saccharides and polysaccharides. The polymer may be a D-PLA copolymer selected from D-PLA-PEG diblock, D-PLA-PEG triblock, D-PLA grafted dextran, D-PLA- PPG diblock, D-PLA-PPG triblock, D-poly esters derived from serine, aspartic acid, tyrosine, glutamic acid and glutamine as described above. The polymer may be a copolymer of D- lactic acid with the corresponding hydroxy acid of serine, tyrosine, lysine and other hydrophilic or hydrophobic amino acids.

Both the polymer and the peptide active agent used in forming a stereocomplex of the invention may comprise a plurality of hydrophilic functionalities that remain intact in the stereocomplex. The hydrophilic functionalities may vary based on the polymer and active agent used. For example, where the polymer is PLA, the hydrophilic functionalities may be carboxyl groups and carboxylic acid groups. Generally speaking, the hydrophilic functionalities may be hydroxyl groups (found in polymers, proteins, peptides, hormones, etc), carbonyl groups (found in polymers, and a variety of active agents having aldehyde or ketone functionalities), carboxyl groups (found in polymers and active agents having carboxylic acids functionalities), amino groups (found in polymers, proteins, peptides and generally in amino acid-containing materials), sulfhydryl groups (found in polymers and active agents in a form of thiols), phosphate groups (found in polymers, proteins, nucleic acids, etc), ether groups, (present in polymers and active agents such as proteins and peptides), ester groups (found in polymers and active agents having e.g., triglyceride functionalities), glycosidic groups (present in polymers having e.g., saccharide functionalities and certain active agents), peptide functionalities and others. The hydrophilic functionalities are oriented outwardly, namely are exposed on the surface of the solid stereocomplex. The surface exposure is achievable by preparing the stereocomplex in water. In some aspects, the invention provides a stereocomplex of a hydrophilic stereoregular biodegradable polyester and a peptide active agent or a protein, the stereocomplex having outwardly extending hydrophilic functional groups.

In some embodiments, the stereocomplex is a solid composite of a polyester and a peptide active agent, wherein the composite is a hydrolysable stereocomplex of the polymer and the peptide active agent; or is formed into a solid composite.

In some embodiments, the stereocomplex is a particulate material having surfaceextending (or surface-exposed) plurality of hydrophilic functionalities, wherein the hydrophilic functionalities are functionalities present on the polymer and/or the peptide active agent.

In some embodiments, the stereocomplex is in a form of a nanoparticle or microparticles.

In some embodiments, the stereocomplex is in a form of a hydrogel with a protein.

In some embodiments, the stereocomplex is a water-dispersible material.

In some embodiments, the stereocomplex is formed in water or in an aqueous medium.

In some embodiments, the stereocomplex is formed by a method comprising combining a solution of the peptide active agent of a given spatial configuration and a solution of the polymer of an opposite spatial configuration under conditions permitting complexation of the polymer and the peptide active agent, wherein one or both of the solutions is an aqueous solution.

In some embodiments, the solution of the polymer is added onto a solution of the peptide active agent.

In some embodiments, the method comprises treating a solution of the peptide active agent of a given spatial configuration with a solution of the polymer of an opposite spatial configuration under conditions permitting complexation of the polymer and the peptide active agent, wherein both the solution comprising the peptide active agent and the solution comprising the polymer are aqueous solutions.

In some embodiments, the weight ratio of the peptide active agent: polymer is between 1 :100 and 100:1, or between 1:1 and 5: 1.

In some embodiments, the polymer/poly ester is a D-configured polyester. In some embodiments, the polyester is a water soluble homopolymer or block copolymer containing at least one stereoregular block that is of the opposite configuration to the L-configured peptide or protein.

In some embodiments, the polyester is a stereoregular polymer having a stereochemical regularity in the sequential repeating units. In some embodiments, the polyester contains a stereoregular block chain of at least 7 monomer units. In some embodiments, the monomer units are selected from D-lactic acid and D-oc-hydroxy acids derived from hydrophilic amino acids or tartaric acid and combinations thereof. In some embodiments, the D-oc-hydroxy acids are derived from serine, glutamine, glutamic acid, lysine, threonine, aspartic acid, cysteine, asparagine, histidine, tyrosine and methionine. In some embodiments, the polyester is a block copolymer of a stereoregular homopolymer and a hydrophilic polymer. In some embodiments, the polyester is a D-polylactic acid (D-PLA).

In some embodiments, the peptide active agent comprises a chain of at least three L- amino acids.

In some embodiments, the peptide active agent is a drug entity or a cosmetically or agriculturally effective amino acid polymer having between 5 and about 50 amino acids.

In some embodiments, the peptide active agent is selected from antibiotic peptides, anticancer peptides, cardiovascular peptides, endocrine peptides, antiviral peptides, antibacterial peptides, antifungal peptides, gastrointestinal peptides, opiate peptides, plant peptides, respiratory peptides, and vaccine peptides.

In some embodiments, the peptide is selected amongst adrenocorticotropic hormone (ACTH), endorphin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormones, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropinreleasing hormone (TRH), and vasopressin and vasoactive intestinal peptide (VIP).

In some embodiments, the stereocomplex comprises (i) a polyester selected from stereoregular D-polylactic acid (D-PLA), D-PLA block copolymers with glycolic acid, racemic lactic acid, caprolactone, ethylene carbonate, polyethylene glycol, poly(2-hydroxy butyrate), and poly(2-hydroxy butyrate) block copolymers; D-stereoregular poly(a-hydroxy esters) or polymers made from tartaric acid should be considered; and (ii) a peptide active agent.

In some embodiments, the polyester and the peptide active agent are chemically associated through an ionic or a covalent bond.

In some embodiments, the stereocomplex is of D-PLA-PEG diblock, D-PLA-PEG triblock, D-PLA grafted dextran, D-PLA-PPG diblock, D-PLA-PPG triblock, or D-poly(oc- hydroxy esters) derived from serine, aspartic acid, tyrosine, glutamic acid and glutamine.

In some embodiments, the stereocomplex comprised a plurality of surface exposed hydrophilic functionalities or hydrophilic functionalities outwardly oriented, said hydrophilic functionalities being hydroxyl groups, carbonyl groups, carboxyl groups, amino groups, sulfhydryl groups, phosphate groups, ether groups, ester groups, glycosidic groups, and peptide functionalities.

The invention further provides a method for forming a stereocomplex of a polymer, as defined herein, and a peptide active agent, the method comprising combining a solution of the active agent of a given spatial configuration (having either D or L configuration) and a solution of the polymer of an opposite spatial configuration (having the other of D and L configuration) under conditions permitting complexation of the polymer and the active agent, wherein one or both of the solutions is an aqueous solution.

In some embodiments, a solution of the polymer is added onto a solution of the peptide active agent.

In some embodiments, the method comprises treating a solution of the active agent of a given spatial configuration (having either D or L configuration) with a solution of the polymer of an opposite spatial configuration (having the other of D and L configuration) under conditions permitting complexation of the polymer and the active agent, wherein one or both of the solutions is an aqueous solution.

In some embodiments, the process comprises providing a solution of the active agent and/or providing a solution of the polymer.

In some embodiments, both solutions (the solution comprising the active agent and the solution comprising the polymer) are aqueous solutions. In some embodiments, the solution comprising the polymer and the solution comprising the active agent are provided each at a concentration (of the polymer and the active agent, respectively) such that when combined, the weight ratio of active agent: polymer may be as disclosed herein, e.g., between 1 :100 and 100: 1 or between 1: 100 and 5: 1.

In some embodiments, both the solution comprising the polymer and the solution comprising the active agent are aqueous solutions further comprising an acid (e.g., trifluoroacetic acid, TFA) and other additives, e.g., metal salts, organic solvents, alcohols, surfactants, etc.

Various salts and metal ions may be present in the stereocomplexation solution to alter the stereocomplex outcome. The ionic strength may affect the degree of complexation and form different shapes and size of particle sizes. Metal ions may complex with hydrophilic components which may affect the accessibility of the polymers towards strereocomplexation. L-PLA block copolymers may be added to the complexation solution to compete with the peptide and loosen the complexation. Aqueous solutions of water miscible solvents such as DMSO, DMF, alcohols, and acids may be added to the complexation solution. Surfactants and water miscible non-ionic molecules such as TWEENs or SPANs as well as mono- and polysaccharides may also be added.

In some embodiments, the aqueous solutions may be acidic or basic, with a pH typically ranging between 2 and 10.

In some embodiments, the conditions permitting complexation include maintaining the combined solutions at a temperature between room temperature (23-32°C) and 100°C. In some embodiments, the combined solutions are maintained under continued mixing or stirring. In some embodiments, the conditions include one of (i) stirring at room temperature (23-32°C); (ii) stirring at a temperature below the boiling point of the medium solvent(s); (iii) stirring at a temperature between room temperature and 100°C.

The polymer and active agent used in a method of the invention is, independently, as disclosed herein. In some embodiments, the polymer is D-PLA. In some embodiments, the active agent is a peptide, as disclosed herein.

The stereocomplex obtained by a method of the invention is any of the stereocomplexes disclosed herein. Thus, in some embodiments, the method comprises combining an aqueous solution of a peptide of a given spatial configuration (e.g., L- configuration) and an aqueous solution of D-LPA under conditions permitting complexation of the peptide polymer and D-LPA.

Stereocomplexes of the invention may be characterized as water-formed or as having exposed hydrophilic functionalities as disclosed herein. In some embodiments, a stereocomplex of the invention is provided that is prepared by a method of the invention. In some embodiments, a stereocomplex of the invention, as disclosed herein, comprises a stereoregular polymer and an active agent such as a peptide, wherein the polymer and the active agent having opposite configurations and wherein the stereocomplex having exposed hydrophilic functionalities; the stereocomplex being formed by a method comprising combining a solution of the active agent of a given spatial configuration (having either D or L configuration) and a solution of the polymer of an opposite spatial configuration (having the other of D and L configuration) under conditions permitting complexation of the polymer and the active agent, wherein one or both of the solutions is an aqueous solution.

In some aspects, the invention further provides a method for forming a stereocomplex of a stereoregular polyester and a peptide active agent, the method comprising combining a solution of the peptide active agent of a given spatial configuration and a solution of the polyester of an opposite spatial configuration under conditions permitting complexation of the polymer and the active agent, wherein one or both of the solutions is an aqueous solution.

In some embodiments, the solution of the polyester is added onto a solution of the peptide active agent.

In some embodiments, the method comprises treating a solution of the active agent of a given spatial configuration with a solution of the polyester of an opposite spatial configuration under conditions permitting complexation of the polyester and the active agent, wherein one or both of the solutions is an aqueous solution. In some embodiments, the method comprises providing a solution of the active agent and/or providing a solution of the polyester.

In some embodiments, the solution comprising the active agent and the solution comprising the polyester are aqueous solutions. In some embodiments, each of the solutions is an aqueous solution maintained at a pH ranging between 2 and 10. In some embodiments, the conditions permitting complexation include maintaining the combined solutions at a temperature between room temperature (23-32°C) and 100°C.

The invention further provides a water-formed solid composite of a water-soluble stereoregular polyester and a peptide active agent, the composite being in a form of a stereocomplex according to the invention, wherein the weight per weight % of peptide active agent-to-stereoregular polyester is between 1 and 80%.

The invention further provides a water-dispersible stereocomplex, as disclosed herein, the stereocomplex being of a water-soluble stereoregular polymer and a water-soluble bioactive peptide or inactive protein; the polymer and the peptide or protein having opposite configurations, wherein the stereocomplex having exposed hydrophilic functionalities; and wherein the stereocomplex is formed in an aqueous medium (being water or containing water).

Excluded from the scope of the present invention stereocomplexes formed in an organic or a non-aqueous medium. Also excluded are processes comprising use of organic or non-aqueous solutions.

The invention further provides a water-formed solid composite or material of a water- soluble polymer and a peptide active agent, the composite/material being in a form of a stereocomplex, wherein the weight per weight % of peptide active agent-to-stereoregular polymer is between 1 and 80%.

The invention further provides a stereocomplex of a water-soluble D-PLA block copolymer and a peptide active agent. In some embodiments, the D-PLA block copolymer is a D-PLA block of at least 7 monomer units conjugated or grafted to a water-soluble polymer such as polyethylene glycol, polypropylene glycol, a peptide or a protein, saccharides and polysaccharides and mixtures thereof.

Despite the fact that stereocomplexes of the invention may be prepared in an aqueous medium, they are nevertheless hydrolyzable. Over time, in presence of water, the interaction between the polymer and the active agent degrades or weakens resulting in the release of the active agent. The degradation of the stereocomplex releases the active agent over time, wherein the rate of release may be governed by proper selection of the polymer. As such, the stereocomplex may be regarded a platform or a system for the slow release or controlled release of the active agent. Alternatively, the stereocomplex may be regarded a prodrug of the active agent, wherein degradation of the interaction between the polymer and the active agent releases the active agent in a form and at a concentration available for absorption by a tissue.

The hydrolysis and release of the active agent may be immediate, or sustained. In some configurations, the release is constant over a period of time. In other configurations, the release is sustained over a period of time. In some cases, the stereocomplex is administered in a formulation further comprising a further active agent, e.g., same or different peptide, which is free or not stereocomplexed. In such cases, the release profile of the active agent may exhibit immediate release (through release of the free active agent) and a subsequent long term or sustained or controlled release of the stereocomplexed active agent over time.

The stereocomplex may be used as is, or in a powder or a particulate form, or may be formulated as an aqueous or a non-aqueous formulation. The invention further provides a formulation comprising a stereocomplex according to the invention. Formulations of the invention may be aqueous formulations or non-aqueous formulations and may comprise one or more additional actives or non-active components which may be provided separately or outside of the stereocomplex. In some embodiments, the stereocomplex may be of a first active agent, e.g., protein, and a second different or same active agent may be provided in the formulation in a free form or in a form that is not a stereocomplex.

In some embodiments, the formulation is configured as a matrix material for controlled release of the active agent. The release may be for medicinal or biomedical purposes, for cosmetic purposes, or for agricultural purposes, or for experimental purposes.

Where the formulation is a pharmaceutical composition, it may be formulated or configured for any mode of administration, including for example oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, rectal, inhalation, nasal and vaginal administration. Alternatively, the formulation may be administered to a mucosal tissue of the oral cavity, nasal cavity, and the lungs. The administration may be for preventing or treating a disease or a condition treatable by the active agent comprised in the stereocomplex. Where the formulation is a non-pharmaceutical composition, its application and use may be according to acceptable protocols in the art. Further provided is a formulation comprising a stereocomplex according to the invention. In some embodiments, the formulation is an aqueous formulation or is configured as a matrix material for controlled release of the peptide active agent. In some embodiments, the formulation is a gel or a hydrogel formulation.

The invention further contemplates a method of delivering an active agent to a cell or a tissue or to an aqueous medium or an aqueous environment or to a subject, the method comprising treating the cell or the tissue or the aqueous medium or the aqueous environment or administering to the subject a stereocomplex or a formulation/composition thereof, wherein the stereocomplex comprises or consists a stereoregular polymer and the active agent and is configured to degrade over time to thereby release the active agent (in or in a vicinity of the cell, or the tissue, or the aqueous medium, or the aqueous environment, or in an organ or tissue of the subject).

The invention further provides a method for administering to a subject an active agent under conditions of controlled release, the method comprising administering to the subject a stereocomplex of the invention or a formulation/composition thereof, wherein the stereocomplex comprises or consists a stereoregular polymer and the active agent and wherein the stereocomplex is configured to degrade over time to thereby controllably release the active agent. In some embodiments, the stereocomplex or formulation thereof is administered via oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, rectal, inhalation, nasal or vaginal administration.

Also provided is a polymeric drug delivery platform for the delivery of a peptide active agent in vivo or ex vivo, the platform being composed of a polymer, e.g., a polyester in a stereocomplex with the peptide active agent, wherein the stereocomplex of the polymer and the peptide active agent is water soluble and hydrolysable to release the peptide active agent over time.

In some embodiments, the drug delivery platform is provided in a protein-based formulation.

In some embodiments the platform is configured for delivery of the peptide active agent over a period of 2 to 100 days. In some embodiments, the drug delivery platform comprises a stereocomplex according to the invention.

In some embodiments, the drug delivery platform is in a form of a gel, a hydrogel or an aqueous solution. In some embodiments, the drug delivery platform is for delivering the peptide active agent in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of nonlimiting example only, with reference to the accompanying drawings, in which:

Fig. 1 provides illustration of a peptide stereocomplex where only the loose protein chains are stereocomplexed, without affecting the 3D protein structure.

Figs. 2A-B provide an illustration of different scenarios of D-PLA/L-peptide stereocomplexation (A) and peptide drug release by DPLA chain scission and detachment for peptide release (B).

Fig. 3 depicts stereocomplexation between D-PLA and L-PLA (top) and hetero- stereocomplexation between D-PLA and L-Peptide (bottom), attributed to similarity in their structure backbone in the context of torsion angles and bond length.

Fig. 4 shows average CD spectra for D-PLA-PEG (red), L-PLA-PEG (blue) and racemic mixture D, L-PLA-PEG (gray) as were recorded in 0.4 cm cells from 190-300 nm with a step size of 1 nm and a bandwidth of 2 nm. D configuration and L configuration present reversed graphs.

Figs. 5A-B are FTIR spectra of stereocomplexes of D-PLA-PEG Insulin at 10, 20 and 30% w/w insulin. Stereocomplexes were prepared in aqueous solution, 60°C for 3 days. Each sample was recorded between 4000-400 cm^{- 1} by 16 scans.

Fig. 6 presents EDx results for insulin, D-PLA-PEG polymer and the 20% insulin/D- PLA-PEG stereocomplex. The polymer only presents peaks of carbon and oxygen with no nitrogen. There is a peak of a nitrogen atom for the insulin and the stereocomplex.

Fig. 7 presents DSC thermograms of PEG-PLA and its stereocomplex with insulin. The stereocomplex was obtained by spontaneous precipitation from water solution, stirring at 60°C for 3 days. DSC are from first run heating at a rate of 10 °C/min. PLA-PAG, presenting one endothermal peak and D-PLA-PEG/Insulin stereocomplex showing a broad peak.

Fig- 8 shows in vitro release of Insulin. insulin-D-PLA-PEG sterecomplexes with different insulin loading. Release studies were performed in phosphate buffer pH 7.4 at 37°C with shaking. Stereocomplexes were prepared in aqueous medium at 60°C for 3 days. Insulin in the releasing medium was determined by uBCA method.

Fig. 9 shows glucose blood levels of Akita mice and non-diabetic mice. D-PLA- PEG/Insulin stereocomplex (17mg) was delivered to mice SC (0.31 ml), once. Blood samples were taken from the tip tail and checked by a glucometer. Blue- Akita mice treated with stereocomplex. Orange- Akita mice with no treatment. Grey- non-diabetic mice given stereocomplex treatment. All represented results are mean+SD^

Fig. 10 depicts synthesis of PEG-PLA and Dextran-PLA with precise PLA chain length.

Fig. 11 provides FTIR spectra of: 20%w/w D-PLA-PEG/Insulin stereocomplex, pure Insulin, D-PLA-PEG polymer; and polymer-insulin (20%w/w) mixture. Each sample was recorded between 4000 and 400 cm-1 by 16 scans.

Fig. 12 provides DSC thermograms of D-PLA-PEG, insulin-polymer mixture, and its stereocomplex with various insulin percentages. DSC was performed at a rate of 10° C min^-1, using 10 mg of each sample.

Fig. 13 provides DSC thermograms of samples at different time points of the stereocomplexation reaction: blue: 20%w/w of insulin and polymer unreacted mixture, yellow: stereocomplex product after 3h of stereocomplexation reaction, greenstereocomplex product after 24 h of stereocomplexation reaction, brown: stereocomplex product after 24h of stereocomplexation reaction. DSC was performed at a rate of 10°C min^-1, using 10 mg of each sample.

Figs. 14A-C provide (a,b) TEM images of D-PLA-PEG/insulin stereocomplex after 72 h of stereocomplexation reaction-presents rounded particles of — 500 nm size, (c) D- PLA-PEG/insulin stereocomplexation reaction mixture, 24 h post complexation initiation- presents a dry mixture of the reaction content that is mostly free or partially stereocomplexed insulin and D-PLA-PEG in the form of needles and nanoparticles.

Figs. 15A-H Atomic Force Microscopy measurement of: A) D-PLA-PEG polymer, no specific structure is observed. B- Insulin presented a common powder-like structure. C) D- PLA-PEG/insulin mixture, 1 minute after mixing, before formation of sterecomplex, powder-like structure is seen. D) stereocomplex 24 h post mixing, interlocked rods are seen. E) D-PLA-PEG/insulin stereocomplex. Defined interlocked rods are observed. F) Focused view of an isolated D-PLA-PEG/insulin stereocomplex. Interlocked, coiled rope-like structure is shown. G) 3D perspective view of D-PLA-PEG/insulin mixture, 1 min after mixing, discovered dispersed powder topography. H) 3D perspective view of D-PLA- PEG/insulin stereocomplex, presented coiled ropes-like interlocked rods arranged into circles. Measurements were made using Bruker Multimode AFM. Scans were made in tapping mode using AC240 probe. Three different areas on the surface of each sample were investigated to ensure a good reproducibility. A scale bar is presented on each individual picture.

Fig. 16 presents insulin content in the supernatant of the reaction media at different time points over the stereocomplexation reaction relative to the insulin content in the reaction media at the initiation of stereocomplexation reaction. Samples were taken from the reaction media at regular time intervals. All samples were centrifuged, and the insulin content in the supernatant was analyzed by a uBCA proteins analysis kit. Red- Insulin/D-PLA-PEG mixture. Blue- Insulin/L-PLA-PEG mixture. Green- Insulin/D,L-PLA-PEG mixture. Blackinsulin alone. The results are of three replicates. All represented results are mean + SD.

Figs. 17A-D are schematic illustrations of homo- and hetero-stereocomplexation (SC), -O- represent ester bond, -N- represent peptide bond (A); PLA hydrolytic degradation and release of the peptide from stereocomplex (B); de-complexation and displacement for peptide release from stereocomplex (C); competitive complexation possibilities (D).

Fig. 18 shows TEM analysis of the insulin and mPEG-PLA with their stereocomplexes. DETAILED DESCRIPTION OF EMBODIMENTS

Peptide activity depends upon their structure and any modification in their chemical/3D structure severely affects their therapeutic efficacy. Furthermore, polymeric drug delivery strategies, where the drug is entrapped in a polymer matrix and released by diffusion, have also had limited success with peptide delivery. Fabrication of micro/nano formulations requires stringent conditions (agitation and use of organic solvents), which affect the peptides.

Polyesters have been extensively investigated in the context of medical applications and drug delivery. Poly(lactic acid) (PLA) and its copolymers with glycolic acid and caprolactone are the most used biodegradable polymers for biomedical applications, including drug delivery, due to its biocompatibility, favorable mechanical properties, and diversity and safety. Its biodegradability is based on the hydrolytic cleavage of ester bonds along the polymer chain, making it an ideal candidate for in vivo applications. Micelles, hydrogels, implants, and scaffolds have been developed based on PLA and copolymers. The PLA building block is lactic acid, an hydroxy acid with a chiral center, similar to amino acids that build peptides and proteins. Thus, polymeric PLA may adopt a three-dimensional structure that is twisted either in a clockwise configuration or counter-clockwise configuration that possess a structural similarity to a peptide structure in terms of torsion angles and bond lengths. Two different molecules with opposite enantiomeric configuration are known to form complexes referred to as stereocomplexes. Previous studies discovered thatD-PLA forms stable stereocomplexes with insulin, LHRH and somatostatin analogs. The three-dimensional molecular “wrapping” of the peptide or protein into the stereocomplex bears tremendous implications for the sustained release of protein drugs. The release of protein drugs depends on the degradation of D-PLA chain interacting with the peptide and the subsequent disruption of the D-PLA/protein complex and not on diffusion of the active peptide through a polymer matrix.

The major advantage of D-PLA stereocomplexation is its potential applicability to almost any peptide or protein drug candidate, as the complexation is affected through the peptide-amide backbone with little influence of the side groups. Since most peptides are water soluble, an aqueous system for stereocomplex preparation is required. As PLA itself is water-insoluble, the conjugation of a water-soluble biocompatible polymer to the PLA chain enhances water solubility and thus the ability to complex a wide range of proteins and peptides. A commonly used water-soluble biocompatible polymer is poly(ethylene glycol) (PEG) that has been used in a wide variety of clinical applications. In terms of sterecomplexes, studies have been made using PLA-PEG block-copolymers, based on complexation of D and L enantiomers of the PLA. This invention presents stereocomplexation in water between a water-soluble D-PLA-PEG and insulin as the complementary enantiomer, to form a water insoluble stereocomplex. Water soluble DPLA- PEG block copolymer with DPLA chain length of about 10 monomer units, required for stereocomplexation was synthesized. Spectral and chemical characterization of the formed stereocomplexes, as well as the in vitro release studies and in vivo activity in lowering glucose blood levels of diabetic mice, is reported. Stereocomplexes with LHRH, semaglutide, somatostatin with D-PLA-PEG were prepared. Water soluble block D-PLA of about 10 units onto saccharides and protein molecules have been synthesized and used for stereocomplexation. Nanoparticle stereocomplexes of 30 to about 500 nanometers are formed by controlled conditions of the stereocomplexation, the ratio of peptide to D-PLA block copolymer and the D-PLA chain length, determines the stereocomplex particle size. Particles that are below 200 nm have potential to cross biological membranes in the GI tract, the lungs and the olfactory for oral absorption, the lungs and nasal spray delivery.

The concept of stereocomplexation is described in Figs. 2 and 3.

Stereocomplexation

The phenomenon of stereocomplexation (Fig. 2A-B) has great implications in material science because the stereocomplexes have superior mechanical and thermal properties as compared to their homochiral polymers. The process of stereocomplexation has been widely studied and best exemplified by the stereocomplex of poly(L-lactide) (L-PLA) and poly(D- lactide) (D-PLA). When L-PLA and D-PLA are mixed together, they form a stereocomplex by packing 0 -form 3i-helices of opposite absolute configuration alternatingly side by side. The resulting stereocomplex shows a melting point 50°C higher than the corresponding crystals of the enantiomers. PLA is a biodegradable polymer that has been extensively studied and utilized for biomedical and pharmaceutical applications.

The stereocomplexation phenomenon is also observed in peptides, with a natural helical structure twisting in a counter-clockwise direction (“L-configured”). Various reports have described stereocomplex formation by peptides. Nahhas et al. reported the formation of a nanosheet stereocomplex system in aqueous solution, where right- and left-handed tripeptide supramolecular hydrogelators are physically mixed without any external stimulus. Sakajiri et al. reported stereocomplex formation between L- and D-enantiomers of poly(g- alkyl glutamate) with short alkyl side-chains (1 to 6 carbon lengths), which resulted in a tetragonal packing -helices. This unusual tetragonal packing symmetry was attributed to the "knobs-into- holes" packing of side chains between L and D helices regularly arrayed in a lattice.

Ueda et al. reported on stereocomplexation between (Sar)m-b-(Leu-Aib)n (m = 2225; n = 7, 8, 10) to design peptide nanotubes of varying diameters and lengths. They also observed that temperature-triggered stereocomplexation leads to fusion of left-handed and right-handed (Sar)25-b-(Leu-Aib)6 (SLL), which led to the formation of vesicles. Ueda et al. also showed that a second-generation polyamideamine (PAMAM) dendrimer bearing right-handed helices to its eight terminals could accommodate eight left-handed helices via stereocomplex formation, generating molecular assemblies with a diameter of 13-14 nm and a thickness of 6 nm. They further observed the formation of nano-sized stereocomplexes with a hydrodynamic diameter in the range of 27 nm when a right- handed third-generation polyamidoamine dendrimer was mixed with left-handed helical peptides. In a recent report, Ueda et al. designed enantiomeric copolypeptides of (Leu-Aib)3-AzF(PSar)-Aib-(Leu- Aib)2, which self-assembled into helical nanotubes by stereocomplexation when the D and L-configured enantiomeric peptide units were mixed together.

Freschini et al reacted 5% LHRH per D-PLA in acetonitrile and obtained a precipitate but they did not find differences between the DSC, FTIR and Raman spectroscopies, and X- ray diffraction data of the precipitate and the starting materials. This can be explained by the low amount of hetero-stereocomplex (<5%), where the analytical methods used were not sensitive enough for detection. Hetero-stereocomplexation: a bioinspired strategy for the delivery of peptide and protein therapeutics

Although the process of stereocomplexation between two polymeric enantiomers has been studied extensively, the process of hetero-stereocomplexation (stereocomplex between polymers with different chemical structures and opposite configurations) is largely unexplored. My research group has been working on hetero-stereocomplexation for over two decades based on the fact that poly -a-hydroxy esters such as PLA share a structural similarity to the backbone of a peptide, in terms of torsion angles and bond lengths (Fig. 3). This similarity arises because, in nature, peptide and protein structures are predominantly composed of L- amino acids as monomer units. Hence, peptides inherently have a 3-D helical structure that twists naturally in a counter-clockwise direction (“L-configured”). While the PLA is composed of repeating lactic acid units, a a -hydroxy acid with a chiral center, peptides are composed of amino acids with a chiral center (Fig. 3). PLA adopts a 3-D structure that is twisted either in a clockwise configuration (D-configured) or counterclockwise configuration (L-configured). Accordingly, when the right-handed helix D-PLA is mixed with the left-handed helix peptide/protein, they form a stable stereocomplex owing to the convex-concave fitness between their surfaces (Figs. 2A-B and Fig. 3).

Previous studies found that D- PLA shows complementarity with the L- configured backbone of peptide moieties such as leuprolide, insulin and. The interaction of PLA and glycine and alanine homopeptides for the induction of PLA crystallization has been reported. The thermodynamic simulations, based on the Flory-Huggins model, predicted that a combination of PLA with amino acids has good compatibility and limited steric hinderance, leading to better nucleation efficiency The implications of this approach are considerable but largely unexplored. First, this kind of interaction is physical, without involving any chemical bond. Thus, the interaction does not affect the chemical structure of the peptide moiety and thereby does not affect its therapeutic activity. Second, the hetero-stereocomplex by this interaction spontaneously forms nano-sized assemblies that range in size from a few nanometers to a few microns and without the need for any specific surfactants or additives. Furthermore, in my previous studies where a precipitate formed spontaneously when the L- peptides (leuprolide, insulin, LHRH, and somatostatin) were mixed with D-PLA in chloroform-ethanol solutions, the formed precipitates showed a controlled release of the peptide drug that depended on the degradation of the D-PLA and the subsequent disruption of the D-PLA/protein complex. The drug release did not depend on the diffusion of the active peptide through the polymer matrix, circumventing the risk of an uncontrolled burst release associated with conventional polymeric carrier systems.

This strategy of hetero-stereocomplexation in aqueous media, provides ample scope for modification in the side-chain of the D-PLA moiety in aqueous media, without affecting the stereo-compatibility of the polymer. Most importantly, the major advantage is its simplicity and universality as it can be used with almost any peptide drug candidate. The switch from organic solvent to complexation in aqueous media is not trivial as peptides and proteins form hydrogen bonds and salts with water molecules and solutes in the water which may affect stereocomplexation. This farther widens the options of forming stereocomplexes of different degree of interactions, depending on solutes preset in the aqueous media: metal and organic salts, pH, stereoregular molecules, surfactants and more. The use of PLA and its copolymers with PEG that are safe and FDA approved materials for invasive drug delivery, reduces the risk of potential toxicity.

Preliminary results of our recent studies showed that the water-soluble D-PLA-PEG block copolymer formed a nano-stereocomplex with insulin in water. The water-soluble block copolymer was made from ring opening polymerization of D-lactide with methoxy- PEG-5000 as initiator to form a copolymer with a PLA chain of about 12 lactic acid units. The complex formed spontaneously when insulin was added to an aqueous solution of D- PLA-PEG. Upon mixing over time, a quantitative precipitate of nanoparticles was produced. The particle size and elemental analysis of the precipitate was determined by a SEM-EDX system which indicates formation of <100 nm and the presence of a peptide content that contain a peptide which released insulin in vitro over a period of 12 weeks. An in vivo study using diabetic mice receiving the long-acting insulin nanoparticles, showed a constant reduction in sugar blood levels for at least 12 weeks. As a control, L-PLA- PEG and D,L- PLA-PEG were reacted with insulin under the same conditions. The racemic polymer did not produce any precipitate while the L- configuration produced some precipitate that released insulin for weeks.

When LHRH was mixed with D-PLA in chloroform-ethanol solution, porous particles released effective LHRH for over one month. When L-PLA was added to the complexation mixture, the yield increased as well as the particle size. The in vivo release was determined by the testosterone levels in mice. When TRH, a tree peptide, was mixed with D-PLA under the same conditions, no precipitate was formed, which indicates that no stereocomplex was formed.

Stereocomplexation of D-PLA with L-PLA and model homopeptides

Synthesis of water-soluble PLA blocks with PEG and dextran of different block chain lengths:

To achieve stereocomplexation in aqueous media, the PLA polymer moiety was functionalized with water-soluble PEG or dextran moiety (Fig. 10).

Briefly, lactic acid oligomers with varying degrees of polymerization was synthesized by ring-opening polymerization of lactide, using 2(2- methoxyethoxy)ethanol and stannous octoate as an initiator and catalyst, respectively. The hydroxyl end group of the lactic acid oligomer was then activated using N,N' -carbonyldiimidazole and coupled to dextran or PEG of varying chain length. The compositions of the grafted PLA blocks were optimized to ensure that the resulting polymer moiety is water- soluble without losing its stereo activity. This optimization involved adjusting the chain length and degree of grafting of the PEG or dextran to achieve optimal water solubility and stereo activity of the polymer. The optimized block copolymer system was used to form stereocomplexes with the peptide and protein in aqueous media.

Stereocomplexation studies on the PLA block copolymers under various conditions:

To establish a baseline, the homo-stereocomplexation of D-PLA with L-PLA and their PEGylated and Dextran functionalized block co-polymers were investigated in water and provided a point of comparison for the subsequent studies. The stereocomplexation was carried out in different aqueous media and reaction conditions. Different variable parameters include chain length and composition of the polymer and peptide moieties, ratio of polymer to peptide, and properties of aqueous media. A response surface methodology employing a Box Behnken design (BBD) was used.

Hetero-stereocomplexation of PLA polymers with model L-mono amino acid peptides:

Hetero-stereocomplexation studies determined the phenomenon of hetero- stereocomplexation and the interaction between different combinations of D-PLA-PEG or dextran block copolymers with homo- peptides of different chain lengths. L-PLA-PEG and D,L-PLA-PEG of similar molecular weights as well as the homo-D-peptides were used as a reference. Custom synthesized mono-amino acid peptides of 3, 6, 9, 12 and 15 amino acids of serine, glutamine and alanine, representing hydrophilic, bulky and hydrophobic amino acid residues, were purchased and used as model homopeptides.

A combination of experimental and simulated data was used to generate a mathematical model that can predict the stereocomplexation between two enantiomeric moieties. Small- and wide-angle X-ray scattering techniques were used to appraise the lattice parameters of the polymer, peptide moieties, and their stereocomplex system. A combination of other techniques such as NMR, spectroscopy, DSC etc. were used to characterize the stereocomplexation. The experimental data were fed into a computer simulation program for molecular modelling to gain valuable insights into the stereocomplexation mechanism.

The kinetics of stereocomplex formation between the different species of D-PLA copolymers and peptides was determined by monitoring the reduction in the starting materials and the increase in precipitate with time. The early stage of stereocomplexation, where the interacting molecules are still in solution, was determined by spectral analysis as. Block copolymers of PLA of different chain lengths and structures, homopeptides and therapeutic peptides at different reaction conditions and mole ratio of starting materials were studied. Competitive studies among stereo-molecules were investigated. For example, mixing D-PLA with L-serine homopeptide in the presence of L-PLA may result in homo- stereocomplex and/or hetero-stereocomplex or/and a mixed stereocomplex of all three stereoregular components.

Degradation and de-complexation studies

Degradation studies: As part of understanding hetero-stereocomplexation, the degradation processes of the PLA chain toward the release of the stereocomplexed peptide were investigated. The release of lactic acid (LA) and water-soluble oligomers, parallel to the release of the peptide from the complexes, were studied. As a reference, detailed degradation studies on the corresponding D-PLA starting material and the stereocomplexes of D-PLA and L-PLA of different chain lengths were performed.

De-complexation studies: To determine the composition of the stereocomplex at any time-point, after preparation, storage at different conditions, and during degradation and release in vitro and in vivo, a de-complexation process was developed where the peptide or protein were released from the stereocomplex by applying conditions that reduced the stereointeractions forces. This was achieved by competitive stereointeraction with excess L-PLA chains or in the presence of surfactants. Such de- complexation studies allowed us to determine the peptide and protein properties, including biological activity after preparation and at any time during storage or degradation studies. Mathematical modeling applied to determine the process of degradation and release of the homopeptides and therapeutic peptides.

Characterization of the developed hetero-stereocomplex systems

The fabricated stereocomplex systems, were evaluated using a combination of experimental and simulated data. A combination of different X ray diffraction (powder diffraction) and state-of-the-art solution scattering (small- and wide-angle) techniques were used to elucidate the stereocomplexation arrangement. The hetero-stereocomplex of polymer and peptide moieties were purified and the lyophilized powder was characterized using powder diffraction to determine the crystal structure and to provide insights into the arrangement of the molecules in the stereocomplex system, including their packing and symmetry. The diffraction pattern was used to extract the information about the size and shape of the crystalline domains within the sample. The Rietveld refinement analysis was used to calculate the degree of stereocomplexation between D-PLA and its peptide/protein counterparts. Wide-angle X-ray scattering (WAXS) was used to extract the information about the crystal structure, orientation, and molecular packing in solution. Small-angle X- ray scattering (SAXS) was used to gain more insights about the global morphology and its nanostructure by calculating the size and shape of the stereocomplex domains, the interparticle spacing, their organization, the domain sizes, and the degree of aggregation in the solution state. The SAXS data were analyzed using a variety of methods, such as Guinier, Porod, and Zimm analyses, and model fitting, using cutting-edge analysis software developed at the Hebrew University, to obtain information about the size, shape, molecular packing, and surface area of the stereocomplexes. SAXS also provided information about the degree of ordering of the stereocomplexes in solution (i.e., domain sizes over which positional correlations are maintained), which can be used to determine the stability of the stereocomplex. The data derived from powder diffraction, WAXS and SAXS experiments were used to understand the underlying molecular mechanism of stereocomplexation between D-PLA and peptides or proteins. In addition, time-resolved SAXS/WAXS experiments provided insights into the kinetics of release and the stability of the complexes.

The molecular weight of the PLA polymers and peptides was determined using gel permeation chromatography (GPC). The GPC was further utilized to evaluate the kinetics of polymer degradation and disruption of the peptide or protein complex in combination with the findings of in vitro release studies.

The stereocomplexation was further studied using SEM with energy- dispersive X-ray analyses (EDX). EDX was employed to determine the elemental composition of the stereocomplex system and will be compared with that of the individual components (Fig. 6). EDX was also be used to monitor the changes in the elemental composition of the stereocomplex system during degradation and stability studies. As seen in Fig. 6, the stereocomplex (middle) contains nitrogen from the insulin and C and O from both components.

The chiral interaction between the polymer and peptide moieties was investigated by measuring the specific optical rotation of the different stereoisomers using polarimetry. The polarimetry was used to evaluate the impact of different functional groups or modifications of D-PLA on its chirality. The SOR measurement was also used to monitor the kinetics of the early stage of the stereocomplexation reaction.

The characterization of the hetero-stereocomplex systems was done in liquid state as well in solid state for the final nano particles. In liquid state, a variety of methods are available to follow the process of complexation. While, for example, SAXS and DLS are able to determine size and perhaps shape of the developing structures, liquid-state high- resolution NMR provides a molecular resolution. This is particularly important since we might face a heterogenous mixture of complexed, partially complexed and non-complexed molecules. Observing the different molecules separately substantially improved the information gained. A variety of approaches can be applied as detailed below.

Differential scanning calorimetry (DSC) is a calorimetric technique for studying the thermal stability, folding, and ligand binding behavior of proteins and their ligands. DSC can measure the melting temperature (Tm) of a protein or complex, which is the temperature at which the sample material undergoes a cooperative transition, for example from the folded to the unfolded state. In the present case, the results can help to understand the stability of the hetero-stereocomplexes.

Isothermal titration calorimetry (ITC) is used to measure the heat released or absorbed during a biomolecular interaction in solution, and thus can provide information on the binding properties and thermodynamics of biomolecular interactions in solution. The binding affinity can be quantified by the dissociation constant (Kd), which is a measure of the concentration of ligand required to achieve half-maximal binding. In addition, ITC can determine the number of binding sites on a protein and also determine the stoichiometry of binding. ITC can provide information on the kinetics of a biomolecular interaction. As an alternative to ITC, surface plasmon resonance provides analogous information.

Circular dichroism (CD) is a spectroscopic technique used to study the secondary structure of chiral molecules such as PLA to a protein. The interaction of PLA to a protein can induce changes in the secondary structures of the molecules, which can be detected by changes in the CD spectrum. NMR-spectroscopy: The chemical shift (line position) is a fundamental parameter in liquid state NMR spectroscopy. It reflects the electronic environment around a nucleus and can provide valuable information about the molecular structure and the molecular dynamics of a sample. By monitoring the changes in the chemical shift over time or under different experimental conditions, NMR can provide information about the kinetics and thermodynamics of conformational changes. By monitoring changes in the chemical shift upon ligand binding, NMR can provide information about the binding affinity and specificity of the ligand, as well as the location and nature of the binding site.

The most straightforward way to gain information about the progress of the complexation process is to monitor the shape of the molecules/complexes over time or under different conditions. The decisive advantage of NMR over, for example SAXS or DLS, is the molecular resolution that permits one to address proteins and polymers separately. Different scenarios such as shown in Fig. 1 can be distinguished. The method of choice is Pulse field gradient (PFG) NMR, which involves the application of magnetic field gradients during NMR experiments, allowing for the measurement of diffusion properties of molecules/complexes in solution. From the diffusion coefficient, information of the molecule size can be obtained using the Einstein- Smoluchowski-equation. In this project, PFG NMR can also provide information about the heterogeneity of a sample, such as the presence of multiple molecular species or the presence of molecular aggregates or clusters. NMR H-D exchange (NMR hydrogen- deuterium exchange) is widely used in protein structure determination and ligand binding studies. However, this requires selective 15N-labelling of the protein. This is a time- consuming procedure and it remains to be seen if it will be necessary to undertake this effort.

Once the complexes have precipitated out of solution and have formed a solid, the selfdiffusion is very likely too slow to be measured in NMR experiments. Hence, we need another molecular probe to investigate the molecular structure. Fortunately, information about molecular and crystalline structure as well as about complexation are available in solid- state from (i) chemical-shift in 13C-MAS-NMR spectra and (ii) dynamic experiments. The chemical shift (line positions in the 13C-MAS-NMR spectrum) in the solid state is also sensitive to the molecular packing: L-PLA, D-PLA and stereocomplexes of L- and D-PLA can well be distinguished. I expect the same for peptides in the uncomplexed and complexed form. The molecular structure has a remarkable effect on the molecular mobility of the polymer and peptide chains. For example, different crystal modification exhibit rather different time constants for the reorientation of molecular bonds, caused by thermal motion. From these data, it should be possible to characterize the status of the molecules in the final nanoparticles.

Compared with solution NMR, the solid-state NMR methodology is more demanding for operative reasons and the need to observe low natural abundance 13C nuclei in the respective samples.

The anticipated hydrogen bonding or ionic interactions between the peptide and polymer moieties on stereocomplexation will be investigated in detail using a combination of Raman infrared and near infrared spectroscopy. Infrared (IR) spectra will be employed to study the secondary structure of the protein or peptide in the stereocomplex system and for probing the kinetic of polymer-peptide conformation changes by analyzing the shift of amide bands in the infrared spectra. Furthermore, near- infrared spectroscopy will be used to provide information on the hydrogen bonding interactions between the polymer and protein molecules. Raman spectroscopy, on the other hand, will be used to provide structural information on the specific functional groups involved in the stereocomplex formation and the intermolecular bonding by detecting changes in vibrational modes of the molecules.

Microscale thermophoresis (MST) will be utilized to investigate the degree of affinity between the polymer and peptide moiety in the stereocomplex system. Briefly, the fluorescently labelled peptide or protein moiety will be titrated with varying concentrations of D-PLA polymer. The binding interaction between the two moieties will cause a change in the thermophoretic mobility of the fluorescently labelled protein or peptide, which will be measured using MST. By measuring the change in fluorescence of a labelled protein or peptide in the presence of D-PLA, the binding affinity between the two can be measured. This technique will be utilized to study the binding affinity of the protein or peptide with the D-PLA polymer and to investigate the effect of different factors such as temperature, pH, and salt concentration on the binding affinity. The stereocomplexation was further characterized by measuring the physical forces, such as van der Waals, electrostatic, hydration, hydrophobic interactions, and steric forces between peptide and D-PLA surfaces, using a combination of surface force apparatus (SFA) and atomic force microscopy (AFM) techniques. For the SFA technique, peptide moieties and D-PLA were physiosorbed or covalently attached to molecularly smooth mica sheets to minimize their roughness. The samples were mounted in the apparatus on transparent silica lenses to measure the interaction force of two surfaces as they come close together and retract. The multiple beam interferometry was used to monitor surface separation and directly measure contact area and observe any surface deformations occurring in the contact zone. The measurements were conducted in different aqueous media, and effects of pH, ionic composition and other reaction conditions on stereocomplex interaction were inferred. Together with these experimental variables, the SFA was used for the direct determinations of the molecular forces and mechanisms that modulate stereocomplexation-mediated binding of peptide or protein with D-PLA. With the SFA, the electrostatic properties of the interaction will be determined between immobilized proteins and D-PLA surface. Previous reports demonstrated that these measurements probe the local - not global - electrostatic properties of protein surfaces, and thus can be used to detect changes in single charged amino acids.

The stereocomplex (SC) interaction between D-PLA and L-Peptide or protein moieties will also be investigated by force spectroscopy using atomic force microscopy (AFM). A state-of-the art methodology was employed to study the strength and nature of the interaction between the stereocomplex forming moieties. The L-Peptide or protein moiety were grafted onto the tip of the AFM cantilever and the D-PLA was grafted onto the substrate surface and vice versa, to make sure that there is no effect by the orientation.

Using a piezo transducer, the D-PLA functionalized cantilever tip was microscopically advanced towards and retracted away from the peptide moiety grafted on the substrate. Deflection of the cantilever in the approach and retraction was detected using a laser-focused detector on the cantilever. The changes in the force of interaction as a function of distance between the two counter moieties and the topography of the stereocomplex were recorded. The degree of affinity between the counter moieties was quantified by measuring the force required for protein pulling to detach the stereocomplex overcoming the attractive van der Waals forces. The images were acquired to assess the helices area, their size and thereby the morphology of the stereocomplex system. The Bell-Evens model was used to extract the kinetic and thermodynamic parameters of the interaction. These methods were applied for the determination of the stereocomplexes, but also for the investigation of the de- complexation, the degradation process of D-PLA and the release of the stereocomplexed peptide.

Computational modelling:

The experimental data were fed into a computer simulation program to generate a mathematical model. The mathematical model was used to predict and design the stereocomplexation reactions.

Molecular modeling software (such as the Schrodinger Materials Science Suite) was utilized to calculate the trajectories of individual atoms and molecules over time based on Newton's laws of motion. Atomistic Molecular Dynamics (MD) simulations were carried out using the Desmond program, an explicit solvent MD package (version 3.1, Desmond Molecular Dynamics System; D. E. Shaw Research, USA, and version 3.1, Maestro- Desmond Interoperability Tools, Schrodinger) with inbuilt optimized potentials for liquid simulation (OPLS 2005) force field. The smooth particle mesh Ewald method will be used for handling long-range coulombic interactions. Once the model is constructed, it was subjected to a series of energy minimization and equilibration steps to ensure that the system is in a stable configuration and has reached a state of thermal equilibrium. After equilibration, molecular dynamic simulations were carried out to generate trajectories of the system over time. The simulations were run to gain insights into the structural and dynamic properties of the stereocomplex system, including the conformational changes, thermodynamic properties, and intermolecular interactions. Analyses of the molecular dynamic trajectories was used to gather information on the structural and dynamic properties of the stereocomplex system, such as the average distance between the polymers, the orientation of the polymer chains relative to each other, and the fluctuations in the conformation of the polymers over time. Design and development of peptide hetero-stereocomplexes

Studies on D-PLA and therapeutic peptide stereocomplexation:

Upon optimization of processing and formulation variables and their characterization, the applicability of the proposed strategy was evaluated by developing a series of peptide and protein stereocomplex formulations. Preliminary studies in my lab revealed that when D-PLA is stirred with L- Peptides in an organic solvent, a nanostructured system is spontaneously generated without the need for any surfactants or additives. Formulations were achieved in aqueous media using water-soluble PEGylated or Dextran functionalized block D-PLA.

Overall, the design and development of peptide hetero-stereocomplexes involved interaction studies, under different conditions, between water-soluble D-PLA copolymers and peptides of different chain lengths. Optimal conditions and characterization methods were applied. The development of the most efficient and effective hetero-stereocomplex formulation is crucial in evaluating the proposed stereocomplexation hypothesis and laid the foundation for further studies on the applicability of the developed formulation for various therapeutic peptides and proteins.

Suitable analytical methods for the quantitative determination of peptide and protein content, including high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS), were developed and validated as per the International Conference on Harmonization (ICH) guidelines.

Design and development of suitable dosage form:

It should be noted that the stereocomplexes intended for oral and nasal spray use should release the peptide in a short time, less than 24 hours, while the extended-release formulation should release the peptide over weeks. To achieve this objective, several options will be applied such as the use of polymers with short D-PLA chains of 4-10 units, additives that induce de-complexation, and increasing the peptide to PLA ratio so that the complexation sites are limited and only partial complexation may apply. Formulation optimization:

Stereocomplexes with D-PLA were assembled and optimized under different conditions i.e., temperature, aqueous solution pH and ionic strength, polymer peptide w/w ratio, and incubation time. Box- Behnken design, a response-surface methodology, was used to optimize the formulation and process variables. The variables were adjusted to three different levels and their effect on dependent variables such as size and zeta of the nanocomplex, stability of therapeutic moieties and stereocomplex system, and drug release behavior. The identified significant parameters were used to optimize and fabricate the suitable dosage form. The final dosage form can be either liquid for injection, lyophilized reconstituted powdered injection, tablet/capsule for oral administration, or a dry powder inhaler (DPI), depending upon the results of stability, safety and pharmacokinetic studies.

In vitro characterization of developed therapeutic protein hetero-stereocomplex delivery systems

The engineered nanocomplexes were undergo comprehensive characterization to determine particle size, drug content and release, polymer degradation process, physical and chemical stability, dispensability, and stability in the pro-nanodispersion formulation. Particle size and zeta potential were determined using dynamic light scattering (DLS) and electrophoretic mobility, respectively. The shape and morphology of the designed stereocomplex system were studied using a combination of scanning electron microscopy (SEM) and AFM techniques. Quantitative estimation of entrapment and loading efficiency were performed by employing validated analytical method specific for the drug.

The drug release mechanism from the stereocomplex was studied in different buffer systems to simulate bio-relevant conditions. Gel Permeation Chromatography (GPC) was utilized to evaluate the kinetics of polymer degradation and disruption of the peptide or protein complex. Various release kinetic models such as zero order, first order, Higuchi model and Korsmeyer-Peppas were applied to in vitro release data to understand the drug release pattern from the developed system. Stability studies:

Stability studies of the dry complexes were conducted to evaluate the physical and chemical stability of the developed formulation under various storage conditions in accordance with International Conference on Harmonization (ICH) guidelines. The stabilityindicating assay method was developed and validated as per ICH guidelines. The samples were stored at different temperatures, including room temperature, refrigerated temperature, and accelerated conditions. Samples were withdrawn at pre-determined time intervals and tested for drug content, impurities, size and zeta potential of nanocomplex etc. The potential dosage forms include a liquid for injection, a lyophilized reconstitute powdered injection, a tablet or capsule for oral administration, or a dry powder for dispersion prior to nasal spray.

In vitro cell biocompatibility:

The developed formulations further underwent in vitro biocompatibility and toxicity studies to determine its suitability for the selected route of administration. The tests included cytotoxicity assays using appropriate cell lines and hemolysis assays to assess potential blood toxicity. Initially, cell viability tests were performed on cell lines such as HEK293, MCF-7, and HUVEC. These cell lines were exposed to various concentrations of the formulation to assess any potential cytotoxic effects. Hemolysis assay and coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) will be considered. Finally, based on the results of these safety studies in corroboration with findings of stability, the route of administration of the developed formulation was decided.

To predict drug absorption upon administration by different routes, several in vitro models were employed such as cell uptake studies, Caco-2 cell permeability assay, Ussing chamber studies, and other relevant assays. To evaluate intestinal permeability, the Caco-2 cell permeability assay was used, which involves measuring the transport of the drug across the Caco-2 cell monolayer. The Ussing chamber studies were conducted to measure the transport of the drug across the intestinal or pulmonary epithelium. These models were simulate the absorption and transport of the developed formulation across the intestinal epithelium and the lung epithelium. The results of these in vitro studies provided valuable insights into the bioavailability of the developed formulation, which were used to optimize the formulation for better absorption and safety.

Formulation studies:

Scale-up procedures for the therapeutic peptides and protein stereocomplexes, were studied. This issue is essential for the science and technology to be developed in this project. Scale-up may require high mixing, cooling/heating cycles, addition or additives, etc., which may affect the activity of the incorporated peptide or protein, peptide distribution within the stereocomplex mass, the release profile, etc.

Pharmacokinetics and safety studies

Pharmacokinetics studies:

Unlike small molecules that can be detected (or their metabolites) in blood and tissues, peptides and proteins are difficult to isolate from tissue or blood and their delivered dose is very small.

Methods for detection of therapeutic peptide protein in serum or tissue may include detection of small molecules that exist in serum as a result of active peptide or protein in the body. Examples include glucose for insulin and testosterone for leuprolide. Another option follows the delivered peptide using an enzyme-linked immunosorbent assay (ELISA). Growth hormones will be determined in serum using the ELISA method (kit 10 1900; Diagnostic Systems Laboratories Inc., USA). TRH will be determined by ELISA using a radiolabeled ligand that can be obtained from DuPont (UK). Somatostatin will also be detected by radioimmunoassay as described previously. Ranibizumab will be determined by a validated ELISA developed at Genentech, Inc. (USA) using recombinant human VEGF165 for capture and a goat anti-human fragment conjugated with horseradish peroxidase (HRP) for detection. Semaglutide will be monitored in blood using a liquid chromatography tandem mass spectroscopy (LC-MS/MS) assay, following precipitation of the plasma proteins (Celerion Switzerland AG, Fehraltorf, Switzerland). The LC-MS/MS assay was previously validated for bioanalysis of plasma samples in the concentration range 0.729-60.8 nmol/L (3.00-250 ng/mL). Successful stereocomplexes of therapeutic peptides and proteins were evaluated in rats to determine the pharmacokinetics and safety profile of the developed formulations. First, the optimized stereocomplex formulations for the different routes of administration, oral, nasal and long-acting subcutaneous injections were selected based on the results of in vitro studies previously performed. The selected formulations were administered to rats via various routes of administration (i.e., subcutaneous, nasal or oral) at their conventional dose amount and adjusted according to the weight of the animals. The control group were treated with the same dose of the drug in their conventional dosage form without the active peptide or protein. Stereocomplexes of a non-active peptide such as degraded short chain gelatin may be used as a reference.

Blood samples were withdrawn at various time points and analyzed for the representing molecules using a suitable reported and validated bioanalytical method to determine the pharmacokinetic profile of the different treatments. To determine the bioavailability, various pharmacokinetic parameters such as maximum plasma concentration (Cmax), area under the curve (AUC), half-life (tl/2), and clearance (CL) will be analyzed using non-compartmental and compartmental pharmacokinetic modelling.

Safety studies:

The safety profile of the developed formulations was evaluated by monitoring the general health, body weight gain or any other signs of toxicity or adverse effects. Blood samples were collected to determine the hematological and biochemical parameters, such as liver enzymes, kidney function, and inflammatory markers. Histological evaluation of the major organs, including liver, kidney, and spleen were also performed to assess any possible organ toxicity. The data collected from the in vivo studies were analyzed statistically using ANOVA to determine the significance of the results of the developed formulations.

Example 1: Stereocomplexes with insulin:

Materials and Methods:

Chemicals, solvents and insulin were purchased from Aldrich-Sigma Israel or from J.T Baker, USA and used without farther purification. Poly(PLA-PEG) synthesis: water soluble PLA-PEG diblock copolymers were prepared by ring-opening polymerization (ROP) of D,L-lactide, D-lactide or L-lactide (PURAC, Holland) with mPEG ( Mw= 5000, Sigma Aldrich, Israel) in the presence of stannous octoate catalyst. A sample synthesis is as follows: 50 pL of a 100 mg mL^-1 solution of stannous octoate in dichloromethane (DCM) was added to a melt of mPEG-5000 (1.51 g, 0.30 mmol), D, L-lactide (408.22 mg, 2.83 mmol), either L-lactide (385.5 mg, 2.67 mmol) or d-lactide (386.35mg, 2.68mmol). Solvent was allowed to evaporate and the vial was purged with dry nitrogen. The mixture was stirred at 120°C for 2h, followed by overnight stirring at 150 °C. The bulk polymer was dissolved in DCM and precipitated into diethyl ether to yield >1.8 g polymer (>95% yield).

The polymers were characterized by proton nuclear magnetic resonance ( ¹ H NMR) (300 MHz, CDCh, 5): 5.15-5.25 (d, LA), 1.5-2.0 (q, LA), 3.55-3.75 (s, PEG);

The formation of an ester bond between mPEG and lactide was determined by IR spectroscopy (2000 LTIR; PerkinElmer, Israel) LTIR 2884, 1754, 1102 cm'¹.

Average molecular weight of the polymers was estimated using Gel permeation chromatography (GPC). Measurements were carried out using a 2695 Waters instrument equipped with Waters 2410 refractive index detector. GPC columns (SHodex, OHpak SB- 803 HQ) were eluted with 0.05M sodium azide aqueous solution at 25°C at a flow rate of 1 ml/min. The molecular weights were calibrated relative to pullulan standards (Sigma Aldrich, Israel).

Circular dichroism (CD) was obtained on the synthesized polymers to observe the dimensional structures differences. Spectra were applied on an MOS-500 spectrophotometer from BioLogic Science Instruments by using 0.05% TFA as a solvent using a polymer concentration of 1 mg/mL. In the near-UV region, CD spectra were recorded in 0.4 cm cells from 190-300 nm with a step size of 1 nm and a bandwidth of 2 nm.

Insulin/PLA-PEG stereocomplex preparation: Insulin solution in 0.05% TFA (10 mg/ml) was added to a D-PLA-PEG diblock copolymer dissolved in 0.05% TFA (9 mg/ml). The complexations were done by adding the polymer solution into the insulin solution, with appropriate volume to reach 5, 10, 20, 30 % w/w of Insulin content in D-PLA stereocomplex. The mixtures were stirred at 350 rpm by magnetic stirrer at 60 °C for three days until precipitation is obtained, allowing for isolation by centrifugation (6500 rpm. 30 min, 7C . 5810 R, Eppendorf, Germany). The precipitate was isolated and dried by lyophilization. L- PLA-PEG and D, L-PLA-PEG were used as controls.

Stereocomplex Characterization: Mass yield was determined by comparing the total mass of the produced dry precipitant to the initial mass of polymer and Insulin. This was used for the complexation.

Concentration of Insulin in the supernatant after centrifugation was quantified by uBCA (CYANAGEN) assay for protein quantification in solution. The absorbance of the measured samples was determined using a UV-visible Plate reader spectrophotometer (BioTek, USA). The absorbance-concentration calibration curve was generated using Insulin standard solutions. uBCA protein analysis: protein assay was carried out under manufacturer’s instructions. Briefly, samples were incubated for 2 h at 37°C, with 150 pL of release sample + 150 pL of the working reagent. Absorbance was then measured at 562 nm using a microplate reader (BioTek, USA).

Complexation rate and turbidity experiment: complexation study was performed as described in Insulin/PLA-PEG stereocomplex preparation. At time intervals (3, 24, 72h), samples of 300ul were removed, and the transmittance (X=430nm) of the sample was measured by UV spectrophotometer (Amersham Biosciences, UK). Afterwards, the sample was centrifuged (6200rpm, 15min), and the insulin content in supernatant was determined by uBCA Protein Assay kit.

Complexation of insulin within PEG-PLA was estimated by Energy dispersive X-ray Analysis (EDX). EDX is an X-ray technique used to identify the elemental composition of the surface materials (up to 1 pm in depth). The EDX instrument with Scanning Electron Microscopy (Quanta 200, FEI Company) was equipped with an EDX detector. Analyses were performed by applying samples which were secured on aluminum stubs using a conductive double-sided tape. They were then sputter coated with palladium at 40 mV for 40 s prior to analysis. The specimens were detected under vacuum (upper limit of 6 x 10-6 mbar) using an EDX detector. Thermal analysis comparing the stereocomplex and its ingredients (polymer and insulin) was performed on Mettler TA 4000-DSC Differential Scanning Calorimetry (DSC), calibrated with zinc and indium standards, at a heating rate of 10°C/min.

Binding and affinity of PEG-PLA to insulin was measured with Micro Scale Thermophoresis (MST). Instrument and settings: Monolith® NT.115^P1C0; MST power = 20%; LED power = 95%. Measurements were carried out in 0.05%v/v TFA and Monolith NT.115 standard capillaries. Binding curves were obtained over a temperature range from 25°C to 45°C at 1 °C increments regulated by the internal temperature control of the Monolith NT.115 Picodevice. Insulin was fluorescence labeled with red-NH2 kit (Thermo Fisher Scientific, USA). The concentration of the labeled Insulin was kept constant at 1 nM and the polymers were diluted in a range from 42,000,000 nM down to 1282 nM. For each temperature, MST measurements were started 120 seconds after reaching the desired temperature.

Size of insulin/PEG-PLA particles were determined using Transmission electron microscopy (TEM). Analysis was performed using JEOL JEM-1400Plus by applying ~10 pL of samples resuspended in DDW to a 200- or 400-mesh copper grid covered by carbon- stabilized Formvar film (SPI, West Chester, PA). The samples were dried overnight before scans were performed at different kV levels. Average size and zeta potential of the polymer and stereocomplex solutions were determined at room temperature by a Dynamic Light Scattering (DLS) instrument (Zetasizer Nano S90; Malvern Instruments, Worcestershire, UK). D-PLA-PEG/insulin particles were added to Phosphate buffer solution (pH 7.4) at concentrations of 5 mg/ml and 1 mg/ml respectively.

In vitro insulin release studies: A phosphate buffer (pH = 7.4) was added to each stereocomplex sample (1 Omg) and kept at 37°C with stirring. The buffer was replaced at time intervals after sedimentation of the particles by centrifugation. All supernatant samples were analyzed for insulin content by uBCA Protein Assay Kit.

In vivo study: 12 weeks old, 16 Akita'^/+ins2 and 5 wiled type (WT) mice were weighted and measured for blood glucose levels. 8 Akita and 5 WT mice were administrated subcutaneously with 17mg of 20% D-PLA-PEG/insulin stereocomplex, suspended in 0.31ml saline. 8 Akita mice did not receive the treatment. The mice were kept with free access to food and water, and were monitored for weight and blood glucose levels twice a week during 14 weeks. At each time point, a blood sample was taken from the tail’s tip and measured twice by a glucometer (Performa, AccuCheck). At the end of the experiment the mice were scarified. Growth necropsy was performed, and the main organs as well the injection sited were visually inspected. The skins of the injection site were submitted for histology evaluation.

Statistical analysis: Analyses of in vivo blood glucose levels were performed with the Microsoft Excel software (2016). The differences between experimental groups were assessed by the paired two-tailed Student's Z-test with significance determined at the 0.05 level.

Results and discussion:

PLA-PEG copolymers:

D-PLA was selected as the polymer for stereocomplex formation due to its complementary stereoregular structure to the insulin chain and its clinical safety record. Since PLA is water-insoluble, conjugation of PLA to PEG, a water-soluble biocompatible polymer-block enhanced water solubility, allowing the PLA solubilize in the insulin aqueous solution. The conjugation of PEG to PLA at an appropriate ratio, grantees the water solubility of the copolymer while allowing PLA chain length of about 11-16 monomer units to allow stereocomplex formation with insulin. De Jong et al. had reported that 2 conformations of PLA exist: 103 -helix with a minimum of 11 monomer units and 3i- helix with a minimum of 7 monomer units. Given that only two helical turns are needed for stereocomplex formation, we synthetized the PEG-PLA diblock copolymers with sufficient length of PEG to ensure water solubility and the minimum amount of repeating lactic acid monomer units required for formation of the helical structure. PEG-PLA is a commercially available for conventional purposes such as stabilizer and delivery systems. However, D- configuration PLA block and PEG with specific block chain length suitable for stereo-interactions in aqueous solution is not available and thus was synthesized. Water soluble D-PLA block of 11-16 lactic acid monomer units, conjugated to methoxy-PEG of MW 5000, was synthesized in 95% yield. The MW and PEG:PLA ratio was confirmed by 'H-NMR. TO determine the PLA chain length, the known integration value of the PEG peak (Peak 1) was compared to the integrations of the lactide peaks (peaks 2,3). Ester bond formation was confirmed by FTIR by the band at 1754 cm'¹. The molecular weight of PEG-PLA was in the range of 6000 as determined by GPC which indicate a PLA chain of 1000 molecular weight which is 10-12 monomer units. For comparison, L-PLA-PEG with similar characteristics was synthesized.

D-PLA and L-PLA are optically active, while the PEG chain is not, so the CD spectra of the D-PLA-PEG should be a mirror view of L-PLA-PEG. Fig. 4 presents the CD spectroscopy chromatogram of the examined polymers. As expected, D and L polymers absorbed at the same wavelengths with the same ellipticity, although with opposite values. The racemic polymer, D, L-PLA-PEG, did not display absorption as it is not optically active.

D-PLA-PEG/Insulin Stereocomplex

Stereocomplexes were formed using two opposite enantiomers. Peptides and proteins naturally adapt a three-dimensional helical structure that twists in a counter-clockwise direction (“L-configured”). Insulin has been widely investigated. The CD spectra of Insulin monomer is a positive in the range of the wavelength we examined, similar to L-PLA-PEG chromatogram, therefore, complexes of Insulin and the enantiomer D-PLA-PEG polymer is a reasonable suggestion.

Stereocomplexes were prepared in aqueous solution. Previous reports dealt with stereocomplexes of D-PLA in dichloromethane-ethanol solution which limited the scope of stereocomplexation to peptides that are soluble in organic solvents such as dichloromethane or chloroform/ethanol mixtures where PLA is soluble. The formation of a PLA-PEG insulin stereocomplex was indicated by the formation of a precipitate in the aqueous solution at 60°C with 5, 10, 20, or 30% w/w insulin per polymer. Precipitates with approximately 60%w/w mass yield were obtained. For comparison, L-PLA-PEG was reacted with insulin under the same conditions and showed precipitate mass yield of only 20%w/w. The reference D-PLA- PEG solution with no insulin or D, L-PLA-PEG with insulin, as well as insulin alone, did not form any precipitate under the same preparation conditions.

Stereocomplexes were analyzed by FTIR, EDx, AFM and DSC. Typical FTIR peptide absorbance at 3290cm^-1 corresponding to amide bonds (N-H), and 1640cm^-1 corresponding to the carbonyl bonds (C = O) were observed. (Fig. 5 and Fig. 11). Comparing FTIR spectra of the stereocomplex and mixture of insulin and polymer at the same mass ratio revealed a shift in the carbonyl bond of the insulin, resulting in a split peak at 1630 and 1661 cm^-1 in the stereocomplex. Additionally, two shifts were observed in the stereocomplex spectrum corresponding to the polymer peaks, which were also present in the mixture, at 1466 cm^-1 (methyl) and 2883 cm^-1 (ester). These results indicate the presence of weak hydrogen bonds between the polymer and insulin chains comprising the stereocomplex system. EDx analysis indicated the presence of nitrogen in pure insulin sample, while, the D-PLA-PEG polymer, did not contain any nitrogen. The nitrogen content of the precipitate was less than pure insulin (Fig. 6), indicating the presence of both protein and polymer in the precipitate. Table 1 provides additional insight by demonstrating the percentages of N, O, and C atoms in the polymer, insulin, and diverse stereocomplex samples, thereby confirming an increase in the percentage of N as the insulin content in the samples increased.

Table 1. N, O, and C percentages as detected by EDx, for D-PLA-PEG, insulin, and stereocomplex of 10%, 20%, and 30% insulin. D-PLA-PEG polymer presented no nitrogen, while Insulin presented 23.4% nitrogen. Stereocomplex system comprised of 5.2, 7.7, and 8.5 percent of nitrogen for 10%, 20%, and 30% Insulin/D-PLA-PEG stereocomplex, respectively.

Thermal analysis is a substation characterization in stereocomplex field because PLA has few crystallinity forms while its stereo interactions creates a new conformational structure, DSC is a technique that measures the thermal behavior of a compound by heat capacity differences, thus it can indicate polymer conformation. DSC results, as shown in Fig. 7 and Fig. 12, revealed distinct thermal behavior of the different samples. L-PLA-PEG and D-PLA-PEG thermograms showed one endothermal peak (59°C), while the stereocomplex system of D-PLA-PEG and insulin showed a very characteristically different peak from the copolymer system alone — a divided endothermal peak. This distinct split in the thermogram indicates diverse regions of changes resulting from new interactions, attributed to the formation of the D-PLA-PEG/insulin stereocomplex. The extent of interaction between insulin and the copolymer system varied depending on the insulin content in the stereocomplex system. Higher insulin content led to broader peaks, with maximums at 54.6 and 48.4 °C observed for a 30% w/w insulin content in the stereocomplex system (Fig. 7 and Fig. 12). Enthalpy values of the sterecomplexes are reduced compared to the D-PLA-PEG polymer itself as the polymer observed 202J/g for the transition state while 72.46J/g and 54.74J/g for 10% and 30% insulin/D-PLA-PEG sterecomplexes, respectively. This phenomenon had been reported as PLA adopts two helical conformations due to stereocomplex formation, while the less stable one is involved in the hetero-stereocomplex of Insulin-D-PLA-PEG. Therefore, as the protein content increased, the enthalpy decreased [12,21], In general, extending or adding a polymer peak in a DSC thermogram indicates diverse sizes of region changes that can be conducted by new interactions. The PLA-PEG peak at around 60°C reflects the long PEG block (5000Da) and the smaller PLA block (~1000Da).

Further to distinctly study the kinetics of the stereocomplexation process, thermograms of the stereocomplex product were recorded at different time points in the stereocomplexation reaction between the copolymer system and insulin. As shown in Fig. 13, the time dependency of the complexation process is demonstrated by the shift in the transition point of the analyzed sample, which gradually moves downward from the characteristic point of basal D-PLA over a period of 72h. These results imply that 72h is required for completion of the stereocomplexation between copolymer system and insulin. These findings were further confirmed with the findings of the formation rate test, examined by insulin content in the reaction media (Fig. 16). The particle size, charge and morphology were determined by DLS, TEM and AFM. TEM analysis confirmed that D-PLA-PEG-Insulin complex generated defined rounded particles of ~500 nm (Figs. 14A-B at two magnifications), while L-PLA-PEG Insulin product was amorphous. D-PLA-PEG/insulin stereocomplex reaction mixture, 24 h post complexation initiation presents a reaction mixture content that is mostly free or partially stereocomplexed insulin and D-PLAPEG in the form of needs and nanoparticles of <200 nanometers (Fig. 14C). DLS analysis confirmed the formation of D-PLAPEG/insulin stereocomplex particle of the size 438 ±65 nm and a Zeta potential of -10 mV. As reference, the same reaction conditions were applied using L-PLA-PEG and D, L-PLA-PEG, of a similar molecular weight and structure. Some minor turbidity was visible after 72 h with the majority of the insulin and the polymer remain intact in the reaction solution. TEM analysis of these reaction solutions, after 72 h, indicated amorphous solid mixture.

Stereocomplex studies utilized AFM measurements, as the stereocomplex exhibited a distinct surface morphology compared to its parent compounds. Here, the measurements were conducted using tapping mode. The tapping mode images give both topography and phase, the latter being a map of energy dissipation that can be related to structural and/or chemical changes on surface (Figs. 15A-H). The results unveiled exclusive topography for the D-PLA-PEG/insulin stereocomplex, forming rough interlocked rods that organized into circular arrangements (Figs. 15D,E,F,H) 3D of these rods revealed a structure of coiled ropes (Fig. 15F). Both insulin by itself and the polymer exhibited a common powder-like structure (Figs. 15A,B,G).

A mixture of insulin and D-PLA-PEG in the same mass ratio, after one minute of mixing, exhibited non-specific topography (Figs. 15C,G). Interestingly, an analysis of the stereocomplex during the preparation phase (24 h post-initiation) revealed the presence of similar interlocked rods, albeit lacking a specific arrangement and being shorter in length (Fig. 15D) This indicates that the stereocomplexation process progresses over time, requiring 3 days for completion. The AFM results align with the aforementioned characteristic outcomes and sizes.

The rate of formation of stereocomplexes was followed for 72 hours by analyzing the free insulin content in the supernatant at various time intervals, following centrifugation. D- PLA-PEG/Insulin mixture, L-PLA-PEG/Insulin mixture, D,L-PLA-PEG/Insulin mixture and pure insulin solutions were investigated (Fig. 16). The results indicate that 3 days are necessary to complete the complexation. For the D-PLA-PEG complexation, the insulin content in the supernatant was less than 10% while for the L-PLA-PEG most insulin remains in solution. Insulin content in pure insulin solutions remained constant during the 3 days of the experiment. These results were corroborated by Micro scale thermophoresis (MST). This method quantifies molecular interactions and binding events based on thermophoresis. The interactions of D-PLA-PEG with insulin and L-PLA-PEG with insulin were examined and revealed different curves that did not overlap, indicating a difference in the interaction of D and L polymer with insulin. The affinity between the polymer and the insulin were not a result of the interactions of the protein with the PEG block, as no interactions were recorded by replacing the enantiomeric PLA-PEG polymer with PEG only. The Kd for insulin/D- PLA-PEG stereocomplex was 4.72 * 105 nM.

L-PLA-PEG polymer may interact with insulin by forming micelles in low yield. However, the insulin content in solution and the amorphous characteristics of the precipitate observed by TEM, indicate an inefficient interaction between L-PLA-PEG and insulin that is different from the -D-PLA-PEG that form a unique stereo-interactions with the insulin.

Each supernatant of the preparation solution was lyophilized, and the solid product was weighted. Then the mass percent of insulin from total solid was calculated for each sample and time point. The weight percent of insulin in the supernatant after lyophilization is the least for D-PLA-PEG/insulin, which lead to the conclusion that most of the insulin content complexed into stereocomplex with D-PLA-PEG. The solid in supernatant after lyophilization is the unreacted solubilized polymer. Another indication for stereocomplex formation is the turbidity of the sample. Turbidity of mixtures during preparation was examined by measuring the transmission reduction vs. time. There was no change in the transmission of pure insulin solutions throughout the experiment, while for solutions of PLA- PEG/insulin mixtures the transmission decreased due to interaction of PLA/PEG with Insulin. Both insulin content in supernatant experiment and turbidity tests confirmed that the stereocomplex formation takes 3 days at 60 °C. In vitro release:

In vitro release of insulin from stereocomplex was determined in buffer pH7.4 at 37°C.The accumulated released percent of insulin was calculated related to the initial amount of insulin in the sample. As seen in Fig. 8, insulin was released for over 85 days, in almost a constant rate with no burst effect, regardless of the insulin content in the stereocomplex. This supports the mechanism of insulin release due to PLA chain degradation. The de- complexation of insulin that is being released should be less dependent on drug loading. These in vitro data are similar to the previously reported insulin release from insulin-D-PLA stereocomplex microspheres prepared in organic solvents.

One of the major advantages of the presented stereocomplex is the absence of burst release. Particularly when used in diabetic patients, a burst release of the insulin could cause for hypoglycemia and must be avoided.

In vivo study:

Akita mouse is a diabetic model for phenotypes associated with type 1 diabetes. This strain carries a dominant mutation in the Mody4 locus on chromosome 7 in the insulin 2 gene which leads substitution of Cys to Tyr amino acid in the chain A eliciting incorrect folding of the insulin protein producing toxicity in pancreatic 13 cells, reduced 13 cell mass and reduced insulin secretion. Akita mice exhibit a low plasma insulin level at 4 weeks of age as well as hyperglycemia. They develop pathophysiological changes related to diabetic complications thus commonly used as an animal model for a variety aspect of diabetics including treatment strategies. PLA-PEG/insulin stereocomplex was examined in the diabetic mice model, to substantiate blood glucose level reduction over a period of 17 weeks. Stereocomplex nanoparticles were injected subcutaneously, and glucose blood levels were determined at certain time points using a glucometer. The basal glucose levels of the Akita mice increased with the age of the mouse until an almost constant level above 500 mg/dL. As shown in Fig. 9, the stereocomplex significantly decreased the mice glucose blood levels. In the first two weeks the glucose reduced to a mean of 200mg/dL, followed by a gradual increase up to about 450mg/dL after 16 weeks. The non-diabetic mice, administered with the stereocomplex, exhibited normal glucose levels throughout the study. Complexing insulin with the complementary enantiomer D-PLA-PEG has revealed a sustained release formulation, consisting high percent of insulin and no organic solvents during preparation. The insulin is protected from degradation since it is interwind with the polymer chain. In vivo findings have confirmed that the stereocomplex is able to release active insulin for 3 months after a single SC injection and reduce blood glucose levels of diabetic mice.

The safety aspects of d-PLA-PEG/insulin stereocomplex for sustained release of insulin followed by subcutaneous administration was evaluated. Although PLA and its copolymers are known as safe and approved for clinical uses, altering composition, size, and structure and incorporation of therapeutic compound require assessment of the product biocompatibility. Examination of mice body weight, growth necropsy, and histopathology findings revealed safety and no side effects. The mice growth was normal and no effects on main organs were observed. Local histopathology of the injection site showed minimal subcutaneous macrophages accumulation (foreign body reaction) related to the degradation of the PLA. This type of reaction is not considered as adverse based on the criteria published by the Society of Toxicological Pathology (STP). No treatment-related changes related to the injection of the insulin were seen. The presented findings add a tier in the safety field of PLA based delivery systems.

Summary: Insulin stereocomplex nanoparticles were prepared by interacting insulin and D-PLA-PEG block copolymer in water at 60°C for 3 days. The nanoparticles were fully characterized and showed in vitro and in vivo continuous release for 14 weeks. An In vivo study was performed on diabetic mice that observed reduced blood glucose levels compared to untreated mice. This study demonstrates the utility of stereocomplexation performed in water, for the preservation and extended release of peptide and proteins, demonstrated on insulin. Since the polymer used, PLA-PEG, is considered as safe and is in clinical use, the safety risks of the stereocomplexes with clinically used peptide and protein drugs, should be low. Moreover, the simple preparation method of precipitation in aqueous media with no organic solvents, and the formation of nanoparticles, provides a major advantage over the known microsphere preparation using organic solvents forming large microspheres. Example 2: Stereocomplexation of insulin with water soluble D-PLA-PEG for use in cultivated meat scaffolds

Aim: Preparation of stereocomplex between D-PLA-PEG block copolymer and different ratios of insulin in aqueous medium.

Materials: mPEG-PLA polymer was prepared as follows: 2.5 g dried mPEG (5 kDa) and 1 g D- lactide was polymerized in the presence of stannous octoate (115 mg in 0.5 mL dried toluene) at 140 °C with stiring for 18 h. The obtained copolymer was purified through dissolving of product in dichloromethane and precipitation in cold diethyl ether. Recombinant Human Insulin was obtained from Biogems (Catalog No. 10-365, Lot #2814123). Deionized double distilled water (DDW) was used.

Methods: a. Preparation of insulin-polymer stereocomplex

Initially the polymer aqueous solution was prepared by heating it in DDW at 85 °C to obtain a transparent solution and cooled to 50 °C. Insulin solution (prepared in pH 2 DDW) was added to the polymer solution and the mixture was kept under stirring for 24 h at 50 °C. After 24 h, the mixture was cooled at room temperature, and then freeze dried. b. HPLC Analysis of insulin

HPLC analysis of the insulin was performed as described below.

Column: Luna C-18(2), 5pm, 150x4.6 mm, 100A Phenomenex, P/No. 00F-4252-E0.

Mobile phase A: 50% Acetonitrile

Mobile phase B: 50% Trifluoroacetic acid aq. Soln. 0.1% (v/v)

Flow rate: 0.5 mL/min

Autosampler temperature: room temperature (~25 °C)

Injection Volume: 10 pL

Column temperature: 35 °C.

UV Monitoring wavelength: 220 nm. CBD Retention time (RT): 9+0.3 min.

HPLC system: MERCK HITACHI HPLC system with INTERFACE D7000, UV DETECTOR L7400, COLUMN OVEN L7360, AUTO SAMPLER L7200 and DEGASSER ERC-3415a.

Standards preparation for HPLC: 1 mg/ml insulin stock solution was prepared in pH 2 DDW. The stock solution was diluted to 10-100 pg/ml in pH 2, DDW.

4. Results: a. Polymer characterization:

(i) Proton nuclear magnetic resonance ('II NMR) spectroscopy

'H NMR spectra were obtained with deuterated chloroform (CDCE) as solvent. ’H NMR spectra of copolymers showed peaks in 5.17 ppm and 1.56 ppm which are attributed to (-CH) and (-CH3) groups of lactic acid repetitive units, respectively. Also, the peaks at 3.38 ppm and 3.64 ppm be also assigned to methoxy end groups (CH3O-) and protons of methylene group (CH2-) in the PEG blocks, respectively. Furthermore, the peaks at 4.3 ppm belong to methylene protons at the end of PEG chains on the site of attachment to methine groups of lactide monomers that the presence of these peaks verified copolymer formation.

(ii) Molecular weight determination

Molecular weight distributions of the mPEG-PLA were determined by Gel permeation chromatography (GPC) equipped with a Waters 1515 Isocratic HPLC Pump, L-7490 Refractive Index detector (RI) (Hitachi), and a Rheodyne (Coatati, CA) injection valve with a 20 pL loop. 5 mg polymer samples were dissolved in 2 mL chloroform. Samples were tested in triplicate. Each sample was filtered through a 0.45pm filter directly into the GPC vials. Samples were eluted with chloroform (HPLC grade) through a linear Styragel column (Waters, MA) at a flow rate of 1 mL/min. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA). Calibration curve and calculations were performed using Empower Software (Murrieta, CA, USA).

From GPC analysis, the weight average molecular weight (M_w), number average molecular weight (M_n) and polydispersity index (PDI) were determined to be 4462 g/mol, 4392 g/mol and 1.02, respectively. (iii) Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra were recorded with a Thermo Scientific FTIR spectrometer (Smart iTR Nicolet iSlO FT-IR) with diamond crystal. The instrument requires ~ 5-10 mg sample that is placed onto a crystal window and spectrum recorded. The scanning range was 400-4000 cm’ ¹ and the resolution was 4 cm’¹. The number of scans for each sample was set to 10.

The diblock copolymer exhibits two characteristic peaks corresponding to each prepolymer. The carbonyl band at 1750 cm’¹ is due to PLA, and the C-H stretching band at 2900-3000 cm’¹ is due to the CH2 group in mPEG. b. Stereocomplex characterization:

Two ratios of stereocomplexation between mPEG-PLA and insulin were prepared and characterized. 1: 10 and 1: 1 ratios of insulin:mPEG-PLA was prepared and checked. The stereocomplex is obtained as a dry powder material.

(i) Scanning electron microscopy (SEM)

SEM of the powder samples was examined. The 1 :10 and 1: 1 insulin: mPEG-PLA stereocmplex did not show any crystals of the insulin.

(ii) Transmission electron microscopy (TEM)

Dispersion of the samples were prepared in DDW and examined under TEM. As shown in Fig. 18, insulin and mPEG-PLA alone looks like aggregated particles. Whereas the stereocomplexes looked like small, well dispersed worm-like structure. For 1: 10 insulin: mPEG-PLA composition, the thickness of the worm-like structure is 15-20 nm. Upon closer look, it appeals that the structure seems to be like interlocking between two structures.

(iii) Differential scanning calorimetry

The thermogram of the insulin and mPEG-PLA polymer was compared with the stereocomplexation with insulin. The endothermic wide peak of insulin that appears at ~80 °C was not seen in the stereocomplex samples.

(vii) Fourier Transform Infrared Spectroscopy (FTIR)

Insulin spectrum show amide bands at (~1640 cm^-1) and (~1540 cm^-1) mainly due C=O stretching vibration, which is characteristic to proteins. These two peaks decrease upon stereocomplexation with the polymer in the ratio 1 :10 and 1: 1, insulin: mPeg-PLA, respectively. Simultaneously, the C=O band for the mPEG-PLA also decreases with increasing amount of the insulin in the stereocomplex.

(iv) HPLC Quantification of stereocomplex

The 1 :10 and 1 : 1 of insulin: mPEG-PLA, respectively, were washed with excess of pH 2 DDW and PBS buffer pH 7, under gentle shaking of 100 rpm for 1 h, at room temperature. The dispersion was then centrifuged at 14000 rpm for 30 mins at 4°C. The supernatant was collected and tested in HPLC for free insulin. Table 2, show the results of the free insulin that was extracted upon washing with the aqueous solutions. Very low or No detection of the insulin indicate a stereocomplex formation.

Table 2: Washing of the stereocomplex in pH 2 DDW and pH 7 PBS solutions. HPLC analysis.

(v) Determination of total insulin in stereocomplex using HPLC

To the known amount of stereocomplex, 200 pL DMSO was added and mixed for 30 minutes until the stereocomplex is completely dissolved. Then pH 2 DDW was added to extract the insulin from the stereocomplex into the aqueous solution. The solution was then centrifuged at 14000 rpm for 30 mins at 4 °C. The supernatant was collected and tested for HPLC for free insulin. Table 3, show the results of the total insulin that was extracted from the stereocomplex. Almost, all of the insulin was recovered from both 1: 10 and 1 :1 insulin: mPEG-PLA stereocomplex.

Table 3: Extraction of insulin from the stereocomplex in pH 2 DDW. HPLC analysis.

c. Release of insulin from the stereocomplex:

The release of the insulin from the stereocomplex was monitored in pH 7 PBS buffer, at 37°C and 100 rpm shaking. In a typical experiment, a known weight of the stereocomplex was taken in 5 mL Eppendorf tubes and to it 5 mL of aqueous solution was added. At each time point 4 mL of the solution was withdrawn and replaced with fresh aqueous medium. Fig. 8 represents the release of insulin from the stereocomplexes in PBS 7.2. A slow release is detected, about 2% per week.

The particles prepared in this Example were incorporated in scaffolds used for cultivated meat production, during its preparation or embedded in the scaffold, after its formation by either swelling or physical entrapment. Insulin is released within the scaffold to reach the cells propagating onto the scaffold.

Example 3: Synthesis of D-PLA block copolymer onto dextran and gelatin

Short chain D-PLA of 8-12 monomer units were synthesized by direct condensation of D-lactic acid or by ring opening polymerization using 2(2-methoxyethoxy) ethanol as alcohol starter. The carboxylic acid terminated D-PLA was activated by N,N’- carboxyidiimidazole, as described in Fig. 10. The PLA chain terminal can be activated also with thionyl chloride or by acetic anhydride. The activated PLA blocks were reacted with dextran in DMF or DMSO where dextran has some solubility to form a water-soluble polymer with grafter D-PLA chains. Similarly, gelatin and short chain proteins were reacted to form water soluble D-PLA-dextran suitable for stereocomplexation. LHRH was stereocomplexed in water to form particles with high yield and constant release of LHRH for 4 weeks. Example 4: Formulation of stereocomplex nanoparticles

Nanocomplexes incorporation in pro-nanodispersion formulation: The peptide-D-PLA stereocomplex nanoparticles were dispersed in water for injection for subcutaneous or intramuscular injection or formulated into pro-nanodispersion formulation for oral delivery. The insulin-D-PLA nanoparticles were dispersed in a typical anhydrous pro-nanodispersion formulation composed of ethyl lactate (400 mg); lecithin (100 mg); Tween 80 (400 mg); coconut oil (200 mg) and Cremophor (200 mg). The formulations was stable and when dispersed in water, it formed nanoparticles of less than 200 nanometer.

Example 4: Preparation of hydrogels of D-PLA-PEG and gelatine

Water soluble D-PLA-PEG copolymer described in Example 2 was mixed at room temperature with gelatine in DDW at a ratio of 1 : 10, 1:5 and 1 :1 in a total solids of 10% in water to form gels of different strength. The more D-PLA-PEG added the stronger gel was obtained. These gels were lyophilized to form scaffolds that can be used for scaffolds for tissue engineering and cultivated meat.

Similarly, gels were prepared when using BSA and plant-based proteins.

A bioactive peptide or small molecule are added to the gel before lyophilization to form a delivery system where the incorporated bioactive agents are being released in a controlled manner, depending on the polymer gel compositions and the preparation method used.

Example 5: Stereocomplexes with water soluble poly(a-hydroxy esters) derived from hydrophilic amino acids

D-configured water-soluble polyesters derived from the amino acids: serine, glutamine, tyrosine and glutamic acid were prepared as previously described in Macromolecules, 2008, 41 (20), pp 7259-7263; Macromolecules (2009), 42(13), 4520-4530. Optically active polymers of molecular weights of Mn~2000 were prepared. Copolymers of these monomers with D-lactic acid were prepared as well as copolymers of the hydroxy derivatives of these amino acids. The polymers were used to form stereocomplexes with insulin, LHRH and somatostatin, as well as gel formation with gelatin and BSA. The bioactive peptides were stereocomplexed in high yields to form nanoparticles and microparticles, depending on the reaction conditions. Gels of different properties were prepared from the stereocomplexation in water at different ratios and reaction conditions.

Claims

CLAIMS:

1. A stereocomplex of a hydrophilic stereoregular biodegradable polyester and a peptide active agent or a protein, the stereocomplex having outwardly extending hydrophilic functional groups.

2. The stereocomplex according to claim 1, being a solid composite of a polyester and a peptide active agent, wherein the composite is a hydrolysable stereocomplex of the polymer and the peptide active agent.

3. The stereocomplex according to claim 1 or 2, being a particulate material having surface-extending (or surface-exposed) plurality of hydrophilic functionalities, wherein the hydrophilic functionalities are functionalities present on the polymer and/or the peptide active agent.

4. The stereocomplex according to any one of the preceding claims, being a water- dispersible material.

5. The stereocomplex according to any one of the preceding claims, formed in water or in an aqueous medium.

6. The stereocomplex according to any one of the preceding claims, formed by a method comprising combining a solution of the peptide active agent of a given spatial configuration and a solution of the polymer of an opposite spatial configuration under conditions permitting complexation of the polymer and the peptide active agent, wherein one or both of the solutions is an aqueous solution.

7. The stereocomplex according to claim 6, wherein a solution of the polymer is added onto a solution of the peptide active agent.

8. The stereocomplex according to claim 6, the method comprises treating a solution of the peptide active agent of a given spatial configuration with a solution of the polymer of an opposite spatial configuration under conditions permitting complexation of the polymer and the peptide active agent, wherein both the solution comprising the peptide active agent and the solution comprising the polymer are aqueous solutions.

9. The stereocomplex according to any one of the preceding claims, wherein the weight ratio of the peptide active agent: polymer is between 1:100 and 100:1, or between 1: 1 and

10. The stereocomplex according to any one of the preceding claims, wherein the polymer is a D-configured polyester.

11. The stereocomplex according to any one of the preceding claims, wherein stereocomplex is in a form of a nanoparticle.

12. The stereocomplex according to any one of the preceding claims, in a form of a hydrogel with a protein.

13. The stereocomplex according to claim 1, wherein the polyester is a water soluble homopolymer or block copolymer containing at least one stereoregular block that is of the opposite configuration to the L-configured peptide or protein.

14. The stereocomplex according to claim 13, wherein the polyester contains a stereoregular block chain of at least 7 monomer units.

15. The stereocomplex according to claim 14, wherein the monomer units are selected from D-lactic acid and D-oc-hydroxy acids derived from hydrophilic amino acids or tartaric acid and combinations thereof.

16. The stereocomplex according to claim 15, wherein the D-oc-hydroxy acids are derived from serine, glutamine, glutamic acid, lysine, threonine, aspartic acid, cysteine, asparagine, histidine, tyrosine and methionine.

17. The stereocomplex according to any one of the preceding claims, wherein the peptide active agent comprises a chain of at least three L-amino acids.

18. The stereocomplex according to any one of the preceding claims, wherein the polyester is a stereoregular polymer having a stereochemical regularity in the sequential repeating units.

19. The stereocomplex according to any one of the preceding claims, wherein the polyester is a block copolymer of a stereoregular homopolymer and a hydrophilic polymer.

20. The stereocomplex according to any one of the preceding claims, wherein the polyester is a D-polylactic acid (D-PLA).

21. The stereocomplex according to any one of the preceding claims, wherein the peptide active agent is a drug entity or a cosmetically or agriculturally effective amino acid polymer having between 5 and about 50 amino acids.

22. The stereocomplex according to any one of the preceding claims, wherein the peptide active agent is selected from antibiotic peptides, anti cancer peptides, cardiovascular peptides, endocrine peptides, antiviral peptides, antibacterial peptides, antifungal peptides, gastrointestinal peptides, opiate peptides, plant peptides, respiratory peptides, and vaccine peptides.

23. The stereocomplex according to any one of the preceding claims, wherein the peptide is selected amongst adrenocorticotropic hormone (ACTH), endorphin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, growth hormones, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), and vasopressin and vasoactive intestinal peptide (VIP).

24. The stereocomplex according to any one of the preceding claims, wherein the stereocomplex comprises (i) a polyester selected from stereoregular D-polylactic acid (D- PLA), D-PLA block copolymers with glycolic acid, racemic lactic acid, caprolactone, ethylene carbonate, polyethylene glycol, poly(2-hydroxy butyrate), and poly(2-hydroxy butyrate) block copolymers; D-stereoregular poly(a-hydroxy esters) or polymers made from tartaric acid should be considered; and (ii) a peptide active agent.

25. The stereocomplex according to any one of the preceding claims, wherein the polyester and the peptide active agent are chemically associated through an ionic or a covalent bond.

26. The stereocomplex according to any one of the preceding claims, wherein the stereocomplex is of D-PLA-PEG diblock, D-PLA-PEG triblock, D-PLA grafted dextran, D- PLA-PPG diblock, D-PLA-PPG triblock, or D-poly(oc-hydroxy esters) derived from serine, aspartic acid, tyrosine, glutamic acid and glutamine.

27. The stereocomplex according to any one of the preceding claims, comprising a plurality of surface exposed hydrophilic functionalities or hydrophilic functionalities outwardly oriented, said hydrophilic functionalities being hydroxyl groups, carbonyl groups, carboxyl groups, amino groups, sulfhydryl groups, phosphate groups, ether groups, ester groups, glycosidic groups, and peptide functionalities.

28. A gel formulation comprising a stereocomplex according to any one of the preceding claims.

29. The gel according to claim 28, being a hydrogel.

30. The gel according to claim 28 or 29, comprising a non-active protein.

31. The gel according to claim 29, wherein the non-active protein is selected from gelatin, collagen, bovine or human serum albumin, plant proteins, fragmented or chopped proteins, and mixtures thereof.

32. The gel according to any one of claims 28 to 31 , configured as cell growing scaffolds, or constructs for drug delivery.

33. A method for forming a stereocomplex of a stereoregular polyester and a peptide active agent, the method comprising combining a solution of the peptide active agent of a given spatial configuration and a solution of the polyester of an opposite spatial configuration under conditions permitting complexation of the polymer and the active agent, wherein one or both of the solutions is an aqueous solution.

34. The method according to claim 33, wherein the solution of the polyester is added onto a solution of the peptide active agent.

35. The method according to claim 33, wherein the method comprises treating a solution of the active agent of a given spatial configuration with a solution of the polyester of an opposite spatial configuration under conditions permitting complexation of the polyester and the active agent, wherein one or both of the solutions is an aqueous solution.

36. The method according to claim 33, wherein the method comprises providing a solution of the active agent and/or providing a solution of the polyester.

37. The method according to claim 33, wherein the solution comprising the active agent and the solution comprising the polyester are aqueous solutions.

38. The method according to claim 33, wherein each of the solutions is an aqueous solution maintained at a pH ranging between 2 and 10.

39. The method according to any one of claims 33 to 38, wherein the conditions permitting complexation include maintaining the combined solutions at a temperature between room temperature (23-32°C) and 100°C.

40. A water-formed solid composite of a water-soluble stereoregular polyester and a peptide active agent, the composite being in a form of a stereocomplex according to any one of claims 1 to 27, wherein the weight per weight % of peptide active agent-to-stereoregular polyester is between 1 and 80%.

41. A formulation comprising a stereocomplex according to any one of claims 1 to 27.

42. The formulation according to claim 41, being an aqueous formulation.

43. The formulation according to claim 41 or 42, configured as a matrix material for controlled release of the peptide active agent.

44. The formulation according to any one of claims 41 to 43, being provided in a gel or a hydrogel form.

45. A method of delivering a peptide active agent to a cell or a tissue or to an aqueous medium or an aqueous environment or to a subject, the method comprising treating the cell or the tissue or the aqueous medium or the aqueous environment or administering to the subject a stereocomplex according to any one of claims 1 to 27 or a formulation thereof, wherein the stereocomplex is configured to degrade over time to thereby release the peptide active agent inside or in a vicinity of the cell, or the tissue, or the aqueous medium, or the aqueous environment, or in an organ or tissue of the subject.

46. A method for administering to a subj ect an active agent under conditions of controlled release, the method comprising administering to the subject a stereocomplex according to any one of claims 1 to 27 or a formulation thereof, wherein the stereocomplex is configured to degrade over time to thereby controllably release the active agent.

47. A polymeric drug delivery platform for the delivery of a peptide active agent in vivo or ex vivo, the platform being composed of a polymer in a stereocomplex with the peptide active agent, wherein the stereocomplex of the polymer and the peptide active agent is water soluble and hydrolysable to release the peptide active agent over time.

48. The drug delivery platform according to claim 47, provided in a protein-based formulation.

49. The drug delivery platform according to claim 47 or 48, for delivery of the peptide active agent over a period of 2 to 100 days.

50. The drug delivery platform according to any one of claims 47 to 49, comprising a stereocomplex according to any one of claims 1 to 27.

51. The drug delivery platform according to any one of claims 47 to 50, in a form of a gel, a hydrogel or an aqueous solution.

52. The drug delivery platform according to any one of claims 47 to 51, for delivering the peptide active agent in vivo.