Polyzwitterionic complexes and methods of use thereof

Abstract

The present teachings relate to polymer systems that include agglomerates or precipitates formed by dipole-charge interactions or dipole-dipole interactions, such as interactions between a polyelectrolyte forming a polyzwitterion complex and a polyzwitterion. The polymer system can be used to deliver encapsulated loadings, such as therapeutic drug products, that release upon elevated ambient pH.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims the benefit of and priority to U.S. patent application No. 63 / 318,760, filed on March 10, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] Coacervates or precipitates formed through dipole-charge interactions or dipole-dipole interactions, such as polyzwitterionic complexes formed from polyelectrolytes and polyzwitterions, can encapsulate and deliver cargo based on changes in the surrounding environment (e.g., pH). Background Art

[0004] The association of two oppositely charged polyelectrolytes can form complex coacervates. Their self-assembly can be driven by the entropic release of counterions and solvents into the bulk mixture. This process can result in a liquid-liquid phase separation between a dense polymer-rich phase and a dilute polymer-poor supernatant. When observed using optical microscopy, the characteristics of coacervation can include the presence of smooth, flowing, and condensed spherical droplets. Complex coacervation can include the formation of bio-inspired monomers with specific three-dimensional structures, the incorporation of charged groups into block copolymers, patterning of order and chirality, and many other physicochemical changes. Complex coacervation can include the association of two polymer chains with uniform negative or positive charges.

[0005] Typically, solutions of coacervates can be stable within a pH range that allows cations and anions to maintain their maximum charge. This phenomenon is illustrated by the top box in Figure 1(a), which represents the range in which coacervates are stable in some polyelectrolyte systems. Outside this range, the ionization of a single chain may not be sufficient to promote coacervation. However, it is also desirable to push this box to a different range, for example, to control retention and release properties, using pH as a chemical trigger. Shifting the box to the left (Figure 1(a), bottom box) to a lower pH range can bring opportunities in the physiological field.

[0006] One example where this situation is advantageous is the gastrointestinal ("GI") tract. Given the large pH gradient between the stomach and intestine (Figure 1(b)), platform technologies that exploit this large gradient to control the retention and release of substances could improve the delivery of cargo to the distal regions of the stomach. This issue is of significant medical interest because many current oral drug delivery technologies are severely degraded upon exposure to the harsh gastric environment. Consequently, many modern therapeutics cannot be formulated for oral delivery and must be delivered intravenously. Since the small intestine is the site of the GI tract with the highest absorption rate and the most efficient delivery, it is tempting to avoid gastric degradation. Furthermore, conditions in the intestine can be much milder due to the production of alkaline secretions that neutralize the luminal pH.

[0007] Therefore, there is a need in the art for technologies that can exploit this important pH difference to deliver drugs, for example, using it as a chemical trigger.

[0008] Furthermore, there is a need in the art for acid-resistant coatings for containers used to store and / or transport acidic liquids. SUMMARY OF THE INVENTION

[0010] Complex coacervation can be viewed as the process by which two oppositely charged polyelectrolytes self-assemble into spherical droplets. In contrast, the present teachings relate to systems and methods comprising polyzwitterionic complexes (or "pZCs") that can be formed by liquid-liquid phase separation of polyzwitterions and polyelectrolytes. Such a system can exhibit orthogonal phase behavior. For example, it can remain intact under acidic conditions but decompose as the pH increases. This process can be controlled by the acid-base balance of the constituent chains. By simulating the gastroduodenal junction, the observed phase behavior can be linked to the physiological conditions within the GI tract. The feasibility of polyzwitterionic coacervates as a pH-triggered payload release technology can be demonstrated using video microscopy. Such a system can address the imminent problem of drug delivery via the oral route and can serve as a packaging solution to improve absorption efficiency.

[0011] The characteristics of pZC make it a drug delivery vehicle that can deliver its cargo based on changes in environmental pH; for example, it can remain intact under low pH conditions such as the stomach but deliver its cargo at higher pH such as the small intestine.

[0012] In one aspect, the present teachings provide polymer systems comprising condensates or precipitates formed by dipole-charge interactions or dipole-dipole interactions. The dipole-charge interactions or dipole-dipole interactions can occur between a polyelectrolyte and a polyzwitterion. For example, a polymer system can include a polyelectrolyte and a polyzwitterion, whereby the polyelectrolyte and the polyzwitterion form a polyzwitterionic complex.

[0013] In some embodiments, the polyzwitterion complex encapsulates a payload (e.g., a therapeutic drug product). The therapeutic drug product can be selected from insulin, probiotics, nonsteroidal anti-inflammatory drugs, vaccines, monoclonal antibodies, and nucleic acids. In certain embodiments, the polyelectrolyte is a pharmaceutically acceptable polyelectrolyte and the polyzwitterion is a pharmaceutically acceptable polyzwitterion.

[0014] In various embodiments, the polyzwitterion is grafted onto a substrate. In such embodiments, the polyzwitterion complex, after formation, can coat the substrate and protect it, for example, from acidic conditions and / or decomposition.

[0015] The polyelectrolyte can be selected from the group consisting of poly(acrylic acid), poly(styrenesulfonic acid), poly(methyl methacrylate), poly(dimethylaminoethyl methacrylate), poly(glutamic acid), poly(aspartic acid), heparan sulfate, hyaluronic acid, and copolymers thereof. The polyzwitterion can be selected from the group consisting of polyzwitterions consisting of phosphorylcholines, sulfobetaines, carboxybetaines, ammonium sulfonates, sulfopyridinium betaine class, cysteine ​​class, and copolymers thereof. In a specific embodiment, the polyzwitterion is poly(2-methacryloyloxyethyl phosphorylcholine). In some embodiments, at least one of the polyelectrolyte and the polyzwitterion is a copolymer.

[0016] In various embodiments, the polymer system further comprises a solvent. The solvent can be water, methanol, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, or a combination thereof. In addition, the solvent can also include one or more of sodium chloride, potassium chloride, sugar, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, and acetic acid.

[0017] In some embodiments, the polymer system is in a solid state. The solid polymer system can include a load. In certain embodiments, the present teachings provide a reconstituted polymer system comprising a polymer system described herein and water.

[0018] The polymer systems or reconstituted polymer systems described herein can form polyzwitterionic complexes at a pH of about 1 to 3.

[0019] In another aspect, the present teachings provide methods for delivering a cargo using a polymer system, wherein the polymer system comprises a polyzwitterionic complex formed from a polyelectrolyte and a polyzwitterion. The polymer system can encapsulate the cargo such that when the pH of the surrounding environment changes, the polyzwitterionic complex dissociates, thereby releasing the encapsulated cargo. In various embodiments, the pH of the surrounding environment is increased to release the encapsulated cargo, such as a therapeutic drug product.

[0020] In some embodiments, methods are provided for producing a polyzwitterion complex layer or coating, wherein the coated substrate includes an acid-resistant layer. In certain embodiments, the polyzwitterion is attached or grafted onto a substrate, which is then immersed in a low pH polyelectrolyte solution to produce the polyzwitterion complex. Such coatings can be used to transport acidic liquids in coated containers or to protect ship hulls from seawater and its contaminants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] It should be understood that the drawings described below are for illustration purposes only. The drawings are not necessarily drawn to scale, with emphasis generally placed on illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.

[0022] Figure 1(a) and Figure 1(b): Schematic illustration of the orthogonal phase behavior of polyzwitterionic complexes and their potential applicability in GI cargo delivery. (a) Comparison of the phase behavior of some polyelectrolyte complexes (top box) with that of polyzwitterionic complexes (bottom box). In some systems, due to the pK a values, there is phase stability (i.e., parameter space in which stable coordination can occur) at intermediate pH values ​​(top box). Maximum coordination occurs when both the positive and negative chains are maximally charged, and typical values ​​for this can include those in the top box. In contrast, for polyzwitterionic complexes, stable spherical droplets can be formed when the pH is in a more acidic range (bottom box). (b) Schematic diagram and design principle of pH variation along the GI tract. In the acidic environment of the stomach, polyzwitterionic complexes can maintain their associated state. When they enter the duodenal space, the pH trace rapidly increases to values ​​of 6-8, which can lead to decoupling.

[0023] Figure 2(a)-Figure 2(f): (a) At low pH (about 2), the repeating units of polyacrylic acid (or pAA) can deionize and act as dipoles, while the phosphoryl groups of the polyzwitterionic repeating units of poly(2-methacryloyloxyethylphosphorylcholine) (or pMPC) can deionize, allowing the monomer to act as a positive charge, resulting in a separation distance r. dc This attraction, combined with the release of counterions, can lead to coordination. (b) Schematic diagram of the electrostatic interaction between pAA and pMPC at a pH of approximately 4. The pAA monomer can be deionized and can act as a dipole. However, the phosphoryl group of pMPC can be ionized, making the monomer electrically neutral and thus also acting as a dipole. The net effect can be a weak dipole-dipole interaction between pAA and pMPC. (c) Due to the pK of the phosphoryl group, a is 2.3, and the charge on the ammonium group is fixed, so when the pH increases from 2 to 4, the net charge Q of the polyzwitterion pZcan vary from +1 to zero. (d) Snapshot of the stacking model of the pAA and pMPC repeat units from Avogadro software. The closest distance between the dipolar pAA and the charged pMPC is 0.38 nm; for the dipolar polyzwitterion, the closest distance is 0.59 nm. (e) Plot of the dipole-charge interaction energy (curve trajectory) for each contact as a function of its separation distance (at pH 2, l B =0.7nm, p1=0.0354, and κ=0.329nm -1 ). The vertical line indicates the closest distance between the charge and the dipole, which is 0.38 nm. (f) The dipole-dipole interaction energy (curve trajectory) of each contact is plotted as a function of its separation distance; at pH 4, l B =0.7nm, p1=0.0354, p2=0.39, κ=0.0329nm -1 The vertical line indicates the closest distance between the dipoles, which is 0.59 nm.

[0024] Figure 3(a) and Figure 3(b): Materials used in the examples: (a) 22 kDa pMPC was synthesized from monomeric MPC and mixed with (b) pAA with a molecular weight of 50 kDa to form a complex.

[0025] Figure 4(a) and Figure 4(b): Stoichiometry of pMPC:pAA at pH 2 (squares) and 3 (circles) plotted against turbidity over the temperature range of 20°C to 40°C. (a) Data from the pH 2 sample show a peak at a mixing ratio of 30:70. In contrast, the data at pH 3, bottom of panel (a), show much lower optical density across a range of stoichiometric ratios. (b) A zoomed-in view of the pH 3 data shown in Figure 4(a), showing a peak at a mixing ratio of 60:40. This optical density peak is much lower but exists over a wider range of stoichiometric ratios. Error bars represent the standard deviation between measurements.

[0026] Figure 5(a) and Figure 5(b): Microscopic appearance of pZC at pH 2 and pH 3. ((a), left) Complex with globular morphology at pH = 2. Images can be taken at 10x magnification using a polarizing objective. This is a representative image corresponding to Figure 6 The letter A in the figure indicates a stoichiometric ratio of pMPC:pAA of 30:70. The scale bar corresponds to a length of 120 μm. This may be similar to some polyelectrolyte complexes. ((b), right) Complex with a globular morphology at pH = 3. Images can be taken at 10x magnification using the same polarizer objective. This is a representative image corresponding to Figure 6In the case of letter B, the stoichiometric ratio of pMPC:pAA is 60:40. The scale bar corresponds to a length of 100 μm. The coordination under these conditions is much less abundant than that seen in (a).

[0027] Figure 6 : Compiled phase diagram comparing mixing stoichiometry (percent pMPC in the total mixture, horizontal axis) versus solution pH (vertical axis), taken from similar Figure 4(a) and 4(b) and the experiments of Figures 5(a) and 5(b). The color axis represents the turbidity value. As determined by optical microscopy, little to no coordination can occur above and beyond the black dashed line. The dark gray area near the letter "A" indicates the highest level of coordination, and the area outside the dashed line indicates that no coordination is detected. The results depicted in this figure are consistent with the results of microscopy. Figure 5(a) is an optical microscopic image of the sample marked with the letter A, and Figure 5(b) is an optical microscopic image of the sample corresponding to the letter "B".

[0028] Figures 7(a) and 7(b): Mechanisms underlying the pH-dependent phase behavior of pMPC-pAA complexes. Dipolar pAA chains are depicted with arrows, and the polyampholyte pMPC is depicted with positive charges along its chains. The corresponding charges for each chain are depicted with negative and positive signs, respectively. In the case of pMPC, charges can be bound to monomers farther from the chain backbone and are therefore depicted as appendages, with negative charges proximal to the backbone and positive charges distal to the backbone. Due to the smaller monomer size and greater chain length of pAA chains, the pAA chain can be more closely approximated as a line of dipoles. ((a), left) pH below 4: Ionization equilibrium can favor the protonation of the pMPC phosphate groups, resulting in a formal charge of +1 for each pMPC monomer. In this case, the exposed positive charges on pMPC interact with dipoles along the pAA chain, promoting coordination and accompanied by counterion release. ((b), right) pH greater than 4: The phosphoryl groups of pMPC can be largely deprotonated, effectively neutralizing the positive charge on the choline groups. In this state, the net charge per pMPC monomer can be zero, and therefore, favorable interactions promoting association with pAA may not exist due to the relatively weak dipole-dipole interactions compared to charge-dipole interactions.

[0029] Figure 8(a)-8(c): Effects of pH and temperature on pZC assembly. (a) Scattered light intensity as a function of solution pH, pMPC:pAA stoichiometry of 50:50. (b) At pH 2, in the sample with a stoichiometric ratio of 30:70 (corresponding to the peak), heating-induced turbidity changes in the aggregates over the temperature range of 20°C–40°C are negligible, demonstrating the temperature stability of the pMPC:pAA complex. (c) The peak sample at pH 3 (pMPC:pAA stoichiometry of 60:40) exhibits similar resistance to thermal fluctuations over the temperature range of 20°C–40°C. Error bars represent the standard deviation between measurements.

[0030] Figure 9 A physical simulation of exposing pZC to physiological conditions relevant to the duodenum. Adding water to the edge of the droplet (top, dots) wets the glass surface along a line near the circumference of the pZC mixture (depicted as a line partially beneath the pZC mixture in the second image below). Next, a small amount of NaOH solution can be micropipetteed into the edge of the previously created wetting line (dots further from the pZC mixture in the third image below). This system, in turn, promotes the orderly, unidirectional, gradual incorporation of NaOH into the polymer mixture.

[0031] Figures 10(a)-10(h): Compilation of video microscopy images of NaOH dissolution of the pMPC-pAA complex. A very small amount of NaOH solution was added next to a drop of polymer mixture (in a sandy area), and the resulting solvent front (also in the sandy area) was recorded using a polarizing objective at 10x magnification. Frames were captured at 2-second intervals using Phantom PCC imaging software and are presented in the order shown in Figures (a) through (h). The scale bar in Figure (a) corresponds to a length of 125 μm and is consistent across the entire frame.

[0032] Figure 11 Figure 2: Absorption spectrum of AlexaFluor-labeled BSA. The absorption peak at approximately 495 nm indicates the presence of fluorescently labeled protein. Note the absence of absorption at 550 nm, which allows the presence of protein and polymer to be tracked separately using absorption spectroscopy.

[0033] Figure 12: The bulk solution of BSA-loaded pZC has the highest absorbance at low pH, which is consistent with previous results. As the pH increases, the pZC assembly can dissolve, so its absorbance drops to baseline levels. The presence of pZC can be monitored by the absorbance at 550nm (diamonds). The absorbance value at 495nm (circles) can be used to track the labeled BSA. As the pH increases and the complex dissolves, the protein absorbance can drop to a baseline level of about 0.2au. This value may be much higher than the baseline reading of the polymer at 550nm, which can be between 0.04 and 0.05au.

[0034] Figure 13 : After centrifugation of BSA-loaded pZC, the absorbance of the supernatant can be measured as a function of pH. Regardless of the measurement, the absorbance at 550nm (squares) can be close to the baseline, indicating that polymer assembly may not be present in the supernatant. As the pH increases, pZC can decompose. At pH 4 or 5, they cannot form complexes, but at pH 2 and 3, the complexes they form can be effectively excluded from the measurement, as indicated by the relatively flat values ​​of the 550nm absorbance measurement results. However, as pZC decomposes, the absorbance value at 495nm (triangles) can steadily increase until it reaches the 0.2au value that can be expected when they are free in solution. (This is the peak absorbance value observed in the initial absorbance curve). Values ​​below 0.2au before pH 5 can indicate that there can be a certain degree of interaction between the polymer chains and the protein under acidic conditions. However, the absorbance increases with increasing pH, which could indicate that BSA preferentially dissociates from the polymer complex at low pH and can be released as the complex dissolves at higher pH values. The complete absence of BSA absorbance at pH 2 could indicate that this cargo interacts closely with its carrier and that the protein cargo can be released into the bulk when the carrier dissolves.

[0035] Figure 14 Figure 2: Fluorescence micrographs of AlexaFluor488-conjugated BSA encapsulated in pZC droplets at pH 2. The images demonstrate preferential segregation of proteins into the polymer-rich phase and their absence in the polymer-poor phase.

[0036] Figure 15: Cross-polarization ("CP") spectrum with a magic angle rotation rate of 7 kHz and a CP time of 1 ms. The 55 ppm peak and the 17 ppm peak are derived from the N-methyl and main chain methyl groups in MPC, respectively. The 180 ppm peak may be derived from the carbonyl signal (present in both PAA and MPC), while the small peaks at 130 and 225 ppm may be rotational sidebands of the carbonyl group. Other peaks may also be assigned appropriately. Main chain methyl groups rather than N-methyl groups may be used to represent the MPC population because the former may have similar molecular dynamics to the PAA carbonyl groups and therefore be excited to a similar degree in the CP experiment. The presence of these peaks indicates that the complex contains both components, pAA and pMPC.

[0037] Figure 16 : NMR spectrum of pMPC and related shifts. 1 H-NMR (500MHz, D2O, δ): 8.05-7.50 (CTA), 4.40-4.00ppm (6H), 3.71ppm (2H), 3.27ppm (9H), 2.20-0.80 (5H+CTA). Detailed Description of the Invention

[0039] The systems and methods disclosed herein can be based on the theory of coordination between charged macromolecules. The two major contributions to intermolecular coordination between polar and charged macromolecules include the electrostatic interaction between all polar and charged repeating units of the coordination chain, and the entropy increase caused by the release of counterions during the coordination process. The coordination between a host macromolecule and a guest macromolecule can be considered. If both the host and the guest are uniformly charged, as in the case of coordination between a polycation and a polyanion, the electrostatic interaction can include a strongly attractive charge-charge interaction, while the release of counterions further enhances this process. On the other hand, if the host is essentially composed of polar repeating units with only a small charge, it can be considered a dipole chain. If the guest is uniformly charged, the electrostatic interaction between the dipole host and the charged guest can include a dipole-charge interaction. In this case, the main driving force from the release of counterions can also promote coordination. If both the host and the guest are dipole chains, the electrostatic interaction energy can include a dipole-dipole interaction, which may be much weaker than a dipole-charge interaction. Furthermore, if both the host and the guest are dipolar, there may be no additional driving force from the release of the counterion. Therefore, under conditions where the dipole-charge interaction is converted to a dipole-dipole interaction, the coacervate complex formed spontaneously by the dipolar and charged chains can be dissolved.

[0040] Thus, the present teachings describe condensates or precipitates formed by dipole-charge interactions and / or dipole-dipole interactions. The condensates or precipitates can include polyelectrolytes and polyzwitterions. For example, the polyelectrolytes and polyzwitterions can form polyzwitterionic complexes (or pZCs).

[0041] definition

[0042] To facilitate understanding of the present invention, certain terms and phrases are defined below.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meanings in chemistry and biotechnology. The chemical structures and formulae described herein are constructed according to standardized valence rules known in the chemical art.

[0044] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0045] In this application, if it is stated that an element or component is included in and / or selected from a list of elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from two or more of the listed elements or components.

[0046] In addition, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the present invention, whether the spirit and scope of the present invention is clear or implicit. For example, when referring to a specific compound, the compound can be used in various embodiments of the present composition and / or the inventive method, unless otherwise understood according to the context. In other words, in this application, the embodiments have been described and depicted in such a way that a clear and concise application can be written and drawn, but it is intended and should be recognized that, without departing from the teachings of the present invention, various combinations or separations of the embodiments can be performed. For example, it should be recognized that all features described and depicted herein can be applied to all aspects of the present invention described and depicted herein.

[0047] It should be understood that, unless the context and usage indicate otherwise, the expression "at least one" includes each of the items mentioned after it individually, as well as various combinations of two or more of the items. Unless the context indicates otherwise, the expression "and / or" should be understood to have the same meaning when referring to three or more items.

[0048] Unless otherwise specifically stated or understood from the context, the use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps.

[0049] Unless otherwise specified, when the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself. Unless otherwise specified or inferred from the context, the term "about" as used herein refers to a variation of ±10%, ±5%, ±3% or ±1% from the nominal value.

[0050] In various places throughout this specification, numerical values ​​are disclosed in groups or ranges. It is specifically intended that the description include each individual subcombination of the members in these groups and ranges. For example, the integers in the range 0-40 are specifically intended to separately disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and the integers in the range 1-20 are specifically intended to separately disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0051] Unless otherwise stated, the use of any and all examples or exemplary language, such as "for example" or "including," is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0052] As used herein, "pharmaceutically acceptable" refers to compounds, molecular entities, compositions, materials, and / or dosage forms that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans (as the case may be). For human administration, formulations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Office of Biologics Standards.

[0053] As used herein, "pharmaceutically acceptable excipients" refers to substances that facilitate administration of the active agent to an individual and facilitate absorption by the individual and that can be included in the compositions of the present invention without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, sodium chloride, normal saline solution, phosphate buffered saline, emulsions (e.g., oil / water emulsions or water / oil emulsions), lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), fatty acid esters, hydroxypropyl methylcellulose, polyvinyl pyrrolidine, and pigments. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents that do not deleteriously react with the compounds of the invention, such as lubricants, preservatives, stabilizers, surfactants (e.g., polysorbate 20 or polysorbate 80), wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings and / or aromatic substances, etc. For examples of excipients and carriers, see Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA (1975).

[0054] Throughout the specification, if compositions and kits are described as having, including, or comprising specific ingredients, or if processes and methods are described as having, including, or comprising specific steps, it is considered that the compositions and kits of the invention consist essentially of, or consist of, the ingredients, and that the processes and methods of the invention consist essentially of, or consist of, the process steps.

[0055] In general, compositions indicating percentages are by weight unless otherwise indicated.

[0056] Polyzwitterionic complexes

[0057] The present teachings provide, in the broadest sense, polymer systems comprising condensates or precipitates formed by dipole-charge interactions or dipole-dipole interactions. Dipole-charge interactions or dipole-dipole interactions can occur between a polyelectrolyte and a polyzwitterion. For example, a polymer system can include a polyelectrolyte and a polyzwitterion, whereby the polyelectrolyte and polyzwitterion form a polyzwitterionic complex.

[0058] At low pH, a polyzwitterion complex is typically formed between a polyelectrolyte and a polyzwitterion. Within the relevant pH range, the polyelectrolyte is typically neutrally charged. Alternatively, a polyzwitterion has a positive charge at low pH, but when the pH increases, it becomes more negatively charged, resulting in the dissociation of the complex formed between the polyelectrolyte and the polyzwitterion. If the complex encapsulates a payload, such as a therapeutic drug product, it can be delivered orally, so that the complex dissociates at the higher pH of the GI tract, thereby avoiding the need to deliver the therapeutic drug product via injection to bypass the stomach. In fact, polyelectrolytes and polyzwitterions form droplets that encapsulate the payload for delivery at low pH.

[0059] In some embodiments, the therapeutic drug product can be selected from the group consisting of diabetes drugs, insulin, probiotics, non-steroidal anti-inflammatory drugs, vaccines, monoclonal antibodies, nucleic acids and combinations thereof. Diabetes drugs can include insulin, glucagon-like peptide-1 (GLP-1) receptor agonists or gastric inhibitory peptide (GIP). Therapeutic drug products are generally drug products that will undergo significant degradation when exposed to the harsh gastric environment. In order to avoid intravenous delivery, the pZC of the present teachings can be used to formulate the therapeutic drug product into an orally deliverable drug product. That is, it is also beneficial to bypass the stomach and deliver the therapeutic drug product in the small intestine because the small intestine is the site with the highest absorption rate and the most effective in the GI tract. Therefore, it should be understood that in certain embodiments, the polyelectrolyte is a pharmaceutically acceptable polyelectrolyte and the polyzwitterion is a pharmaceutically acceptable polyzwitterion.

[0060] In some embodiments, for example, after forming the complex encapsulating the load, the polymer system is formed into a solid state or in a solid state form by freeze-drying the pZC-rich layer. The solid polymer system can include the load, thereby allowing the therapeutic drug product to be stored in a solid form for direct delivery to the stomach with water. In certain embodiments, the solid polymer system is reconstituted with water and optionally includes a pharmaceutically acceptable acid or other pharmaceutically acceptable excipient as needed. In this case, the pH of the reconstituted polymer system described herein can be about 1 to about 3.5, or about 2 to about 3, thereby forming a polyzwitterionic complex.

[0061] In some embodiments, when making a pZC of the present teachings, the pH of the solution can be about 0.5 to about 5, about 1 to about 4, about 1 to about 3, about 2 to about 3, or about 2.5 to about 3.5.

[0062] When it is not solid or forms a pZC comprising a therapeutic drug product, the polymer system also includes a solvent. The solvent can be water, methanol, ethanol, ethylene glycol, propylene glycol, polyethylene glycol or a combination thereof. In addition, the solvent can also include one or more of sodium chloride (NaCl), potassium chloride (KCl), sugar, hydrochloric acid (HCl), sulfuric acid, nitric acid, hydrobromic acid and acetic acid (AcOH). For the delivery of therapeutic drugs, pharmaceutically acceptable excipients such as sodium chloride, potassium chloride, sugar, hydrochloric acid and acetic acid can be used.

[0063] The polyelectrolyte can be selected from the group consisting of poly(acrylic acid) or pAA, poly(styrenesulfonic acid), poly(methyl methacrylate), poly(dimethylaminoethyl methacrylate), poly(glutamic acid), poly(aspartic acid), heparan sulfate, hyaluronic acid, and copolymers thereof. In some embodiments, the polyelectrolyte is pAA.

[0064] The polyzwitterion can be selected from the group consisting of phosphorylcholines, sulfobetaines, carboxybetaines, ammonium sulfonates, thiopyridinium betaines, cysteine ​​and copolymers thereof. In a specific embodiment, the polyzwitterion is poly(2-methacryloyloxyethylphosphorylcholine) or pMPC.

[0065] In some embodiments, at least one of the polyelectrolyte and the polyzwitterion is a copolymer.

[0066] In various embodiments, polyzwitterions are grafted onto a substrate. In such embodiments, the substrate can then be contacted with a polyelectrolyte solution, for example, by immersing it in a polyelectrolyte solution at a low pH (e.g., 0.5-5), to form a polyzwitterion complex and a robust, acid-resistant layer on the substrate. These layers can be used to coat the interior of containers used to transport acidic liquids. Alternatively, these layers can be applied to ship hulls to protect them from seawater and related contaminants.

[0067] For example, consider a polymer system designed for oral delivery of an acid-sensitive therapeutic drug product where the host molecule is poly(acrylic acid) (pK a '6.2), the guest molecule is a polyzwitterion pMPC (phosphoryl group pK a'2.3). Under the experimental conditions of interest for delivering therapeutic drugs through the stomach (e.g., pH less than 4), polyacrylic acid can be considered a dipole chain. When the pH is less than 4, the polyzwitterion can essentially be a polycation, while when the pH is greater than 4, it can be a dipole chain. Since dipole-charge interactions accompanied by counterion release can lead to coordination, while the strength of simple dipole-dipole interactions may not be sufficient to lead to coordination, a mechanism can be envisioned in which the polyzwitterion repeating unit of the guest is charged at lower pH and dipolar at higher pH, thereby designing a transition from a coordinated state to a non-coordinated state within the aforementioned pH range.

[0068] The energetics associated with the coordination of condensates involving polyzwitterions can be difficult to calculate quantitatively due to the rich chemical details and the lack of knowledge of the local dielectric constants that mediate the various electrostatic interactions in the system. However, the energetics of the specific system described above can be qualitatively and heuristically illustrated as follows. Figures 2(a) and 2(b) schematically illustrate the situation at low and high pH, ​​respectively, centered around the neighborhood of a pair of complementary repeat units. At the low and high pH considered here, the acrylic repeat unit can be considered to have a dipole moment However, at low pH, the polyzwitterionic repeating unit can carry a net positive charge due to the protonation of the phosphoryl groups; at higher pH, the polyzwitterionic repeating unit can have a dipole moment. The net charge of the MPC group (Q pz ) as a function of pH is shown in Figure 2(c). Therefore, at low pH, the electrostatic energy of coordination can include the dipole-charge interaction energy (v dc ), at higher pH the electrostatic energy of coordination can include dipole-dipole interaction energy (v dd ). If we average over all allowed orientations of dipoles spaced r apart, then v dc (r) and v dd (r) can be given by:

[0069]

[0070] and

[0071]

[0072] where k B T is the Boltzmann constant multiplied by the absolute temperature, l B is the Bjerum length (=e 2 / (4π∈0∈k B T), e is the electron charge, E0 is the vacuum permittivity, is the effective dielectric constant of the medium), and κ is the inverse Debye length.

[0073] Figure 2(d) shows a model of the closest distances between the acrylic acid dipole and the charged ammonium group, and between the acrylic acid group and the dipolar polyzwitterion, based on Avogadro software. Using the MMFF-94 (Merck Molecular Force Field) algorithm, the interaction energy can be minimized from many starting configurations, and the calculated geometric configurations can be close to the global minimum. It is estimated that these distances can be 0.38 nm and 0.59 nm, respectively. B =0.75nm (equivalent to aqueous solution at room temperature), κ =0.329nm at pH 2 -1 , at pH 4, κ = 0.0329 nm -1 , p1 / e = 0.0354 nm (equivalent to a dipole moment of 1.7 D for acrylic acid), and the dipole moment of the dipolar polyzwitterion is estimated to be 0.39 e (equivalent to 18.74 D), then Figures 2(e) and 2(f) show the v calculated according to equations (1) and (2). dc and v dd Here, according to Figure 2(d)v dc and v dd The cutoff distances are 0.38nm and 0.59nm respectively.

[0074] As can be seen in Figures 2(e) and 2(f), the pairwise interaction energy of charge and dipole is an order of magnitude stronger than that of a pair of dipoles. The latter can be much weaker to achieve any coordination at room temperature. For the former coordination process involving charged groups, the release of counterions from the associated monomers may provide an additional significant driving force. Therefore, coordination is expected at lower pH values ​​less than 4, while no coordination is expected at higher pH values ​​greater than 4. While the theoretical demonstration presented above may be conceptual and does not account in great detail for subtleties such as water reorganization and the local dielectric constant, it can provide a reasonable guide to the observed phenomena outlined here. It is important to note that during the coordination process, the amine groups of the MPC may displace protons from the acrylic monomers, forming ion pairs in the coordinated state. In this case, the stronger charge-charge interaction (compared to dipole-charge) and the increased translational entropy of the released protons may result in a more stable complex. That is, variables can include the pH-modulated charge / dipole moments of the host and guest repeat units, the ionic strength, and the polarizability of the medium. With the above concepts in mind, functional polyzwitterionic systems can be designed by selecting certain properties of these materials.

[0075] It may be necessary to protect the cargo delivered into the GI tract. Some relevant parameters may include the pH of the stomach (which is close to 2) and the pH of the duodenum (which increases to values ​​of 5-8). In addition, the temperature of the GI tract may be maintained at 35-40°C. With these physiological parameters in mind, it is anticipated that various functional chemical agents will be useful in this context.

[0076] Taking advantage of this natural guidance, pMPC and pAA can be used to explore the feasibility of this new class of materials, comprising interacting chains of polyzwitterionic and anionic polymers, for their suitability for retaining and releasing cargo within the GI tract. Furthermore, pAA has a simple structure and widespread availability, and is also a common food additive approved by the U.S. Food and Drug Administration. To survive the harsh conditions of the stomach, it can be coordinated at low pH and dissociated at higher pH.

[0077] In addition to this pH-dependent behavior, the complexes remain intact over a wide temperature range, encompassing ambient conditions found in the laboratory or home environment, as well as physiologically relevant conditions exceeding 35°C. These complex challenges can present obstacles in the design of oral drug delivery systems, as most drugs rapidly disintegrate upon exposure to gastric contents, reducing their absorption efficiency. Consequently, blood concentrations of the administered drug may be lower than the recommended ingested dose. For very expensive drugs, or those that cause acute toxicity when local concentrations are high enough, simply increasing the dose is not an acceptable solution. For these reasons, drug packaging designed specifically for the small intestine (particularly the duodenum) could be a frontier in pharmaceutical research. If a payload does reach the small intestine, unperturbed by the gastric environment, the payload can be delivered at a much higher blood concentration than would typically be the case. Furthermore, if it were possible to protect sensitive payloads from gastric corrosion, new opportunities would emerge for packaging advanced therapeutics. Example

[0078] The systems and methods disclosed herein involve the formulation of polymer systems that undergo liquid-liquid phase separation and self-assemble into droplets. These polymer systems, such as pZC droplets, represent a new class of materials that are distinct from other condensates because their composition differs from other oppositely charged polyelectrolytes. It is demonstrated that the behavior of pZC droplets under low pH conditions disrupts most electrostatic assembly, nonlinear stoichiometric and pH-dependent behavior, and globular morphology. Next, a mechanism is demonstrated that explains this phenomenon and links these observations to applications in gastrointestinal physiology. Finally, methods are described for controlling, exploiting, and observing the nonequilibrium response characteristics of these materials in real time using video microscopy under conditions that mimic relevant aspects of the duodenal environment. pZCs may meet key requirements for gastrointestinal drug delivery and provide a blueprint for strategies to enhance drug delivery to the gastrointestinal tract.

[0079] 1. Materials and Synthesis

[0080] Monomeric MPC was purchased from Sigma Aldrich (MilliporeSigma, Burlington, MA, USA) (Figure 3(a)). 1 g of 2-methacryloyloxyethyl phosphorylcholine (MPC) was dissolved in 3 mL of deionized water. 19 mg of 4-cyano-4-(phenylthiocarbonylthio) valeric acid dissolved in 0.4 mL of methanol and 0.1 mL of a 20 mg / mL stock solution of 4,4'-azobis(4-cyanovaleric acid) (ACVA) in methanol were then added. The mixture was stirred for 2 h under ice-cooling with N 2(g) The reaction mixture was degassed by purging for 30 minutes. The solution was heated at 70° C. for 6 hours and then quenched by rapidly cooling with liquid nitrogen and opening the reaction vessel for ventilation. The crude product was purified by reverse water dialysis and then lyophilized to obtain the pure product as a light pink solid in 86% yield with a molecular weight (M n ) was 22,000 g / mol. Molecular weight analysis (M) was performed via gel permeation chromatography (GPC) against PMMA calibration standards using an Agilent 1200 series system equipped with a degasser, RI detector, a PFG guard column (8 x 50 mm) and a PFG analytical linear M column (8 x 300 mm, 7 mm particle size) from Polymer Standards Service and an isocratic pump. n 、M w and D). The eluent was 2,2,2-trifluoroethanol (TFE) containing 0.02 M sodium trifluoroacetate at a flow rate of 1 mL / min at 40 °C. 1 H-NMR studies (500 MHz, D2O, δ) revealed the following shifts: 8.05-7.50 (CTA), 4.40-4.00 ppm (6H), 3.71 ppm (2H), 3.27 ppm (9H), 2.20-0.80 (5H+CTA). Figure 16 As shown. pAA (Figure 3(b)) was purchased from Polysciences, Inc. (CAS# 00627-250, Warrington, Pennsylvania, USA) as a 25 wt.% solution and diluted with MilliQ water to prepare a 1 wt.% stock solution. Its molecular weight can be 50,000 g / mol and can be used after dilution without further modification or purification. HCl (aqueous) and NaOH (aqueous) were used to adjust the pH of the stock solution.

[0081] 2. Preparation of pAA and pMPC Samples

[0082] pAA and pMPC stock solutions were prepared by dissolving dry polymer powder (pMPC) or concentrated polymer solution (pAA) in Milli-Q water (resistivity of 18.2 MOhm cm at 25°C, MilliporeSigma, Burlington, MA, USA). The concentration of the stock solutions was set to 1 weight percent. The pH of the stock solutions was adjusted to a range of 2-12 using small volumes of concentrated hydrochloric acid or NaOH in increments of 1 pH unit. The pAA and pMPC stock solutions were mixed in the specified stoichiometric ratio in 10% increments by pipetting them into a microcentrifuge tube containing a certain volume of Milli-Q water (Thermo Fisher Scientific, Waltham, MA, USA) to achieve a final total polymer concentration of 0.75 wt.%. pMPC was added before pAA, and this mixing order was maintained throughout the measurements. Between each step, the sample was vortexed at high speed for 10 seconds to ensure thorough and consistent mixing.

[0083] 3. Optical density

[0084] The results were obtained by using a Tecan Infinite 200Pro instrument (Tecan Group Ltd, Switzerland) measured the optical density or turbidity or total scattered light intensity of each sample.

[0085] Optical density according to the formula Determination, where I 。 is the light intensity not attenuated by the sample, and I is the light intensity transmitted through the sample. Therefore, a reading of T = 0.35 means that the sample causes a 10% attenuation of the light intensity. 0.35 In other words, only 44.7% (100÷2.24) of the I incident on the sample 。 Light reaches the detector. Measurements are made by placing the samples in a clear, flat-bottomed polystyrene 96-well plate (Corning Inc., Corning, NY, USA) and scanning with 550 nm light, as the samples do not absorb light at this wavelength. As a final step, each sample is shaken in a plate reader before measurement to ensure uniform mixing of the batch. Turbidity measurements are performed in 5°C increments over a temperature range of 20°C to 40°C.

[0086] 4. Optical microscopy

[0087] Optical micrographs of a small aliquot of each sample were recorded on a Leica DM 2700P instrument (Leica Camera AG, Wetzlar, Germany) equipped with a single polarizer. Samples were examined at 10x magnification on Fisherbrand glass slides (Thermo Fisher Scientific, Waltham, MA, USA). Still image data were edited and displayed using Phantom PCC 3.5 software (AMETEK Inc. Vision Research, Wayne, NJ, USA), and video micrographs were edited and processed using Phantom Video Player software from the same vendor.

[0088] 5. Dissociation and Video Microscopy of Synthesized On-Glass Mimic Complexes

[0089] In order to create conditions for the controlled and unidirectional application of a basic medium to a solution of a polyzwitterion-polyanion complex, a device was constructed to demonstrate the dissociation process of the complex. Using a micropipette, a relatively large droplet (300 μL) containing pZC was placed in the center of a glass slide and allowed to relax for a few minutes. Next, a much smaller volume of Milli-Q water (15 μL) was added to the edge of the droplet, allowing the two liquids to come into contact and mix. The pipette tip was carefully placed at the intersection of the droplet and the glass slide, and a sliding motion was initiated while depressing the piston to create fine water lines on the glass. This ensured that the glass slide was wetted at a specific location, allowing the subsequent addition of another aqueous solution without surface tension preventing uniform mixing from occurring.

[0090] Next, a similarly small (15 μL) droplet of concentrated NaOH solution was added to the apex of the streak left by the pipette tip when water was previously deposited onto the glass surface. After the addition of NaOH, the surface energy mismatch between the glass slide and the concentrated NaOH aqueous solution is large enough to prevent further wetting of the glass. Therefore, NaOH spontaneously mixes with the polyzwitterion-polyanion solution in an orderly, unidirectional manner, passing only through this channel. Using video microscopy, the progression of the basic medium through the droplet and the subsequent dissociation of the complex at the solution front were monitored by carefully positioning the glass slide under a polarizing objective lens so that the velocity vector of the dissociation front intersected the optical path of the observation area.

[0091] 6. Titration of pAA and pMPC

[0092] 400 μL of each salt-free polymer solution was prepared by diluting the stock solution to a concentration of 0.75 wt.% and then added to a 2 mL glass vial with a small Teflon stir bar. A pH probe was inserted into the solution, with the top of the bulb positioned above the bottom of the glass vial to provide enough space for the stir bar to thoroughly mix the solution. The titrant was manually added directly to the polymer solution in 1 μL increments using a micropipette, allowing sufficient time (1 minute for each titrant addition) for the solution to equilibrate before recording the pH value.

[0093] 7. Coordination of Protein Loading in pZC Droplets

[0094] Conjugated with fluorescent dyes (Alexa Fluor TM Powdered bovine serum albumin (" BSA ") was purchased from Invitrogen (Life Technologies Corporation, Eugene, Oregon, USA). Protein was used as it was when purchased and dissolved in water. After optimizing loading conditions and concentration, BSA solution was added between pMPC and pAA components in the solution to ensure that complexes were formed in the presence of protein. Load absorption studies were performed by simultaneously performing independent optical density measurements at the protein peak absorption wavelength (495nm) and the peak absorption wavelength (550nm, as previously described in the methods section) corresponding to the complex turbidity measurement results. The solution was divided into a polymer-rich phase and a poor polymer phase using centrifugation, and the preferential separation of load was tracked by assessing the lack of protein in the upper poor polymer phase. (The increase in protein concentration in the poor polymer phase can indicate a decrease in coordination level). Confirmatory microscopic images were taken using a Nikon CrestV2 confocal fluorescence microscope (Nikon Instruments, Inc., Melville, New York, USA).

[0095] A. Liquid-Liquid Phase Separation in Polyzwitterion-Polyelectrolyte Solutions

[0096] To evaluate the phase behavior of poly(2-methacryloyloxyethylphosphorylcholine)poly(acrylic acid) (structure shown in FIG2 , abbreviated as “pMPC-pAA”) droplets, the effect of the mixing ratio of the two components on the turbidity of the resulting solution was tested.

[0097] Stock solutions of pMPC and pAA were mixed together at a range of different stoichiometric ratios. Turbidity measurements at defined pH values ​​are plotted in Figures 4(a) and 4(b). At pH 2, a clear, sharp peak (Figure 4(a)) corresponds to a pMPC:pAA stoichiometry of 30:70, indicating that under these conditions, an excess of carboxylic acid is required to interact with a given amount of polyzwitterion. This could mean that relatively less pAA monomer is available for coordination compared to pMPC. Interestingly, at pH 2, a secondary peak appears at 80:20. At pH 3 (Figure 4(b), which is a magnified view of the trace result at the bottom of Figure 4(a)), the peak position shifts to the right, corresponding to a pMPC:pAA stoichiometry of 60:40. This peak is much broader than the peak at pH 2, suggesting that the pH 3 sample is less sensitive to mixing stoichiometry.

[0098] This finding suggests that pMPC's availability for coordination is reduced at pH 3, as fewer pAA chains are available to interact with pMPC under these conditions to produce peak turbidity. Furthermore, the relative heights of the turbidity plots suggest that more coordination occurs at pH 2 than at pH 3. Quantitatively, it is helpful to note that the relative height difference between the peak turbidity at pH 2 (30:70 ratio) and pH 3 (60:40) is approximately an order of magnitude. A cursory comparison of the two micrographs in Figures 5(a) and 5(b) indicates that the number of droplets in the pH 2 sample far exceeds that observed in the pH 3 sample, consistent with the turbidimetric results. Furthermore, the presence of a pH unit difference suggests an order of magnitude difference in proton concentration, a result that is fully consistent with the view that the observed phenomenon is the result of equilibrium in the ionization of the constituent chains. At pH 4 or above, coordination is completely diminished; indeed, micrographs under these conditions suggest a complete absence of coordination. These results can be compiled and summarized in Figure 6 The phase diagram shown.

[0099] A question that may arise from the results so far is whether the droplets in Figure 5 contain both pMPC and pAA, or whether one of the solution components undergoes self-coordination. To address this question, care can be taken to ensure that both polymers participate in droplet formation. First, a pure pMPC solution is subjected to increasing concentrations of hydrochloric acid. The turbidity does not change as the solution pH decreases. The turbidity of pAA alone is insensitive to pH changes. pH-induced self-coordination cannot occur between these two solution components.

[0100] Next, the presence of both pMPC and pAA in the polymer-rich phase can be confirmed, a necessary consequence of the absence of self-coordination. After preparing a representative solution (for example, using a peak pMPC:pAA mixing ratio of 30:70), the polymer-rich phase is spun in a centrifuge and the polymer-poor supernatant is removed. After lyophilizing the solvent from the remaining sample, NMR measurements are performed to detect the presence of each polymer. Evidence of both pMPC and pAA is visible in the resulting spectrum, confirming that both chains are participants in the coordination process described herein. Figure 15 These results are summarized.

[0101] Next, to demonstrate that the properties of the polyzwitterionic coordination are sensitive to changes in pH and stoichiometry (as is the case for some polyelectrolyte-coordinated condensed systems), optical micrographs were systematically recorded over a range of stoichiometric ratios and pH values. Figure 6 There is a clear agreement between the phase diagram drawn in and the qualitative appearance of the samples under microscopic examination. When coordination occurs, it is characterized by the presence of highly dispersed, mobile spherical droplets with length scales ranging from submicrometers to tens of micrometers.

[0102] 5( a ) and 5 ( b ) may include two representative optical micrographs of polyzwitterion complexes taken from samples having peak turbidity values ​​at pH 2 and 3. Figure 6 Labels A and B in the image correspond to the two samples: the 30:70 sample at pH 2 and the 60:40 sample at pH 3, respectively. At pH 2, coordination is present in nearly all samples, while at pH 3, only some samples exhibit spherical droplets near the peak pH. These findings reinforce the results from turbidimetry: the ratio at which coordination is observed is also the ratio at which the turbidity value is highest. Furthermore, when comparing the pH 3 image with the pH 2 image, less coordination is present in the pH 3 sample, regardless of the stoichiometric ratio. This finding is consistent with the correspondingly lower maximum turbidity at pH 3 than at pH 2, as the total scattered light intensity responds to both the size and number of scatterers present. While comparing the number of droplets between the two images is relatively easy, obtaining reliable measurements of droplet size is challenging for several reasons. Most importantly, while droplets can form immediately after mixing, they can also coalesce over time, and the kinetics of this process can be influenced by many factors. Sizing techniques could be significantly improved to measure the R of chains in these droplets. g and R hEqual amounts were observed. Droplet size estimates were obtained by measuring the droplets using ImageJ software. The pH 2 sample contained a population of droplets with a very broad distribution of length scales centered around 2.1 microns, while the pH 3 sample contained far fewer droplets with a narrower length scale distribution centered around 1.65 microns. Finally, at pH 4, no clear evidence of coordination was observed at any stoichiometric ratio. Thus, there is multiple physical evidence that the phase behavior of complexes formed by pMPC and pAA can respond to their chemical environment, as expected from other polyelectrolyte condensates.

[0103] These findings, namely the nonmonotonic dependence of turbidity on the stoichiometric ratio between the two polymers and the presence of abundant arrays of spherical droplets of various sizes in many samples, demonstrate that at sufficiently low pH, pMPC in combination with pAA forms a new class of materials similar to the family of coordinated condensates. Furthermore, their phase behavior is orthogonal to that of conventional polyelectrolyte complexes. In other words, at low pH, pMPC can be stable, but under less acidic conditions, the polymers can no longer interact, leading to decomposition. This behavior contrasts with that of other polyelectrolyte condensates, where coordination occurs only at intermediate pH and decomposes at very low and high pH values ​​(Figure (1)). The mechanism of this orthogonal phase behavior, depicted in Figures 7(a) and 7(b), and its origin in polyzwitterionic compositions, will be described in the following sections.

[0104] B. Mechanistic Basis for the Orthogonal Phase Behavior of Polyzwitterionic Condensates

[0105] To understand the phase behavior of pMPC-pAA complexes, the proton association / dissociation balance of each polymer can be considered. This balance determines the total charge available on each polymer chain, which in turn controls the availability of each polymer for coordination with potential partners.

[0106] In the case of pAA, each chain can be negatively charged in the deprotonated state or neutral in the protonated state. The number of chains in either state can be determined by the pK of the acidic group. a Decision, pK a It has been measured to be 6.2. For pMPC, the presence of two charged groups in each monomer can complicate this balance. The choline moiety is positively charged, and because it is fully quaternized, it does not undergo typical acid-base reactions. Therefore, the choline moiety can carry a permanent positive charge. On the other hand, the phosphoryl group can accept or release a proton depending on the pH of the solution.

[0107] When the pMPC phospho group is deprotonated, its negative charge can neutralize the positively charged choline group. In this state, the monomer has an effective charge of zero. However, when the phospho group is protonated, its negative charge is masked by the choline group. Therefore, the monomer under these conditions has a formal charge of +1. In solutions that promote the protonation of pMPC, the positively charged choline group can interact with any available pAA chain. The specific pH range for the ionization of pMPC can be determined by the pK value of its phosphate group. a Decision, pK a It has been measured to be 2.3.

[0108] Taken together, these results from turbidity measurements and microscopy explain the potential for increased coordination. Using the Henderson-Hasselbach equation, the proportion of positively charged pMPC approaches 80% at pH 2. The proportion of positively charged pMPC monomers decreases with increasing pH, falling to 28% at pH 2.5, 3.8% at pH 3, and so on. This trend suggests that decreasing pH leads to increased coordination. As pH decreases, more pMPC chains in a positively charged state are available for interaction with pAA in solution.

[0109] Based on the data, the mechanism described here can explain the higher intensity and greater number of scattered light observed for the complexes at pH 2 compared to pH 3. By observing the ratio of charged pMPC groups at pH 2 and pH 3 and factoring in the peak sample stoichiometry, the peak turbidity can differ by an order of magnitude between the two pH values. At pH 2, 80% of the monomers are positively charged. Taking 30% of these (the peak stoichiometry of pMPC), 24% of the sample can be composed of charged pMPC units. At pH 3, the peak stoichiometry can be 60%, which, given the 3.8% proportion of charged pMPC monomers, means that 2.3% of the total sample is composed of charged pMPC units. This order of magnitude difference (24% vs. 2.3%) can be closely correlated with the difference in peak turbidity values ​​between the pH 2 and pH 3 experiments. Furthermore, the reduced charging of pMPC at higher pH values ​​can explain the rightward shift of the stoichiometric peak from 30:70 at pH 2 to 60:40 at pH 3. Fewer charged groups on the pMPC means fewer pAA chains are required for interaction. Figures 7(a) and 7(b) conceptually summarize these findings, where Figure 7(a) shows a polyzwitterionic complex 10 formed from polyelectrolyte 20 and polyzwitterion 30. Figure 7(b) shows the polyzwitterionic complex split into its components, polyelectrolyte 20 and polyzwitterion.

[0110] The specific nature of the interaction between pMPC and pAA can be determined using the energetics of charge-dipole interactions. When MPC is at low pH, it can behave as a cation, and its interaction with acrylic acid can be modeled as a charge-dipole interaction. When the ambient pH rises to 4 or above, MPC can become dipolar, while the acrylic acid groups can also remain dipolar. Therefore, under these higher pH conditions, the energetics can be modeled using dipole-dipole interactions. As shown in Figures 2(e) and 2(f), the latter interaction can be almost an order of magnitude weaker. Therefore, a stable complex cannot be formed unless the pH is sufficiently low.

[0111] The coordination behavior of the system is most strongly dependent on the ionization of the pMPC component. While the above explanation is self-consistent, another subtlety can be added to more fully understand the data. The polyelectrolyte coordination process can be driven by the release of counterions into the bulk solution. The interpretation of the coordination mechanism in this section has so far focused on electrostatic principles, primarily related to enthalpic parameters. These considerations can be important for understanding how the chains appear when they associate and what conditions they can adopt in order to participate in coordination in the first place. To understand why they associate, this information can be used to describe the entropic contribution to the phase separation process.

[0112] Initially, the counter-anions bound to the positively charged amine groups on the polyzwitterions can be bound, preventing these small ions from fully exploiting their conversion entropy in solution. However, when the two chains approach each other, the complementary monomer units can interact with each other instead of with their respective counter-ions, allowing the previously associated counter-ions to freely explore the bulk solution. The coordination of each pair of monomer units results in an increase in the entropy of the small ions per association. This can lead to a net increase in the entropy of the system. It can be said that this reaction can be roughly summarized as follows:

[0113] pMPC—POH–N + X - + HOOC—pAA → pMPC—POH–N + HOOC-pAA + X - (3)

[0114] As the reaction proceeds in the forward direction, X - Ions can be released into solution.

[0115] This observation allows us to derive consequences for the mechanism described in this paper. As long as the pMPC can maintain a net positive charge, coordination can occur. However, if the pMPC is a pure dipolar chain with zero net charge, coordination may be undesirable and may not occur (Not Applicable). Writing the reaction as before:

[0116] pMPC-PO - –N + + X - + HOOC—pAA → N / A (4)

[0117] When pMPC is in a neutral dipole configuration, there may be no prospect of phase separation. There may be nothing to drive the above reaction forward. pMPC may be uncharged, so it will not release negative counterions if brought into close proximity with pAA monomers.

[0118] The aforementioned enhancement of electrostatic attraction by the entropy-dependent behavior of counterions and chains is common in condensed matter. The counterion release hypothesis suggests that this mechanism could be the driving force for coordination between polyelectrolytes. Furthermore, extending the concept of ion release as a driving force for the formation of polyzwitterionic complexes is consistent with existing evidence and suggests that this mechanism may have a generalizable generality in driving the phase behavior of all types of charged polymers.

[0119] C. Suitability of physiological temperature and pH standards

[0120] This system is suitable for use in a biomedical setting. It can be tested against three criteria selected based on the physiology of the GI tract. First, the system should remain intact at low pH. Second, it should be unstable at medium to high pH values. The latter criterion accounts for the duodenal environment, where the duodenal gland (Brunner's gland) promotes the secretion of alkaline fluids that neutralize the acidity of chyme entering the stomach (Figure 1(b)). Finally, the complex should remain intact over a wide temperature range, including that of the GI tract. Figures 8(a)-8(c) demonstrate that the pMPC-pAA complex meets all three criteria. In Figure 8(a), high scattered light intensity readings are observed at low pH, indicating stability within the gastric range. A sharp drop in scattered light intensity is observed when the pH reaches 4. Therefore, coordination readily occurs at gastric pH, followed by dissociation at the higher pH of the duodenal space.

[0121] To meet the third criterion, the temperature stability of pZC was examined by selecting the highest turbidity peak from the chemometric study in Figure 4(a) and varying the temperature of each sample from 20°C to 40°C in 5°C increments. The results in Figures 8(b) and 8(c) demonstrate that the polyzwitterionic complexes meet the third criterion—temperature does not strongly affect the coordination level between pZC and pAA. These complexes are largely resistant to fluctuations in their thermal environment, a potential requirement given the potential for materials used in GI tract environments to experience large temperature changes as they enter and pass through the human body.

[0122] To test the nonequilibrium responsiveness of the pMPC-pAA complex to variable pH, a device was constructed that allowed the polymer to be exposed to alkaline solutions in a controlled manner ( Figure 9 (See the "Methods" section for details). This approach is designed to mimic the effects of an alkaline buffer secreted by the duodenal gland. When an alkaline solution is added to a large droplet containing a pMPC-pAA mixture, it flows progressively from one side of the droplet to the other. Figures 10(a)-10(h) summarize video evidence of pH-induced phase instability in pZC solutions. As NaOH moves from right to left, it dissolves the polymer droplets in its wake (the sandy, rough areas). Individual droplets appear to temporarily swell and break apart until they are completely decomplexed. Control experiments using pure water instead of the NaOH solution did not show any such response of the complex to the addition of water, demonstrating that this effect is not due to dilution alone but rather to an increase in the ambient pH. Interestingly, the pH-dependent dissociation is reversible: the complex can be dissolved upon addition of NaOH to the pZC solution. If hydrochloric acid is subsequently titrated into the same solution, the pZC droplets can be restored.

[0123] The experiments in Figures 10(a)-10(h) can be compared with Figure 6 The two experiments differ when considering the distinction between equilibrium and nonequilibrium behavior with respect to pH. Figure 6 The measurements in

[10] were performed on 11 separate samples prepared over a range of discrete and static pH values, while Figures 10(a) to 10(h) describe the dissolution of a single sample whose pH was initially set to 2 and then raised by exposure to an alkaline solution. In other words, Figure 6While providing equilibrium information for the pMPC-pAA complex, Figures 10(a)-10(h) demonstrate that the spherical polyzwitterionic complex can exhibit nonequilibrium (pH-dependent) and responsive properties when exposed to different physiologically relevant conditions. The activity shown in Figures 10(a)-10(h)—the dissolution of the complex after exposure to alkaline media—underscores the responsive nature of the polyzwitterionic complex and suggests its potential use as a GI drug delivery platform.

[0124] After establishing the aforementioned range of activities, cargo can be encapsulated into pZC droplets. For this purpose, the fluorescently labeled protein BSA (bovine serum albumin) was used. The protein was added between the addition of pZC and pAA, allowing droplet formation to occur in the presence of BSA. Solutions prepared in this manner will appear turbid, as before, with a slight color corresponding to the fluorophore. The ternary solutions were spun in a centrifuge to assess whether the protein colocalized with the polymer. Optical density measurements of the supernatant (at 495 nm, the absorption wavelength of the fluorophore) revealed a complete absence of protein in the polymer-poor phase under low pH conditions. In contrast, when labeled BSA alone was centrifuged under the same centrifugation conditions, the absorbance spectrum of the conjugated protein in solution was clearly visible, indicating that the protein did not simply sink to the bottom of the tube with the polymer during centrifugation. To assess whether the polyzwitterion carrier could release its cargo, the experiment was repeated with varying pH. Absorbance measurements of the supernatant clearly showed an increase in the concentration of BSA in the upper phase as the pH increased from 2 to 5. Thus, cargo release is pH-dependent, as expected based on the carrier's solubility as a function of pH.Finally, fluorescence microscopy directly demonstrates protein localization into pZC droplets. Figure 11-15 These results are summarized.

[0125] Coordination between pMPC and pAA is possible. These complexes display many of the same properties observed in polyelectrolyte condensates, even though one of the components is a polyzwitterion rather than a polyelectrolyte. The presence of the characteristic globular assemblies observed in condensate solutions was confirmed. Their sensitivity to the chemical environment and mixing stoichiometry was demonstrated. Furthermore, orthogonal phase behavior arising from the asymmetric charging of the two constituent polymers was demonstrated. A mechanism explaining this behavior was also delineated. Finally, these properties were exploited to demonstrate the potential applicability of polyzwitterionic condensates as a platform technology for pH-triggered cargo release.

[0126] One implication of the mechanism described here is that the specific acid-base chemistry of monomers, particularly polyzwitterionic monomers, can determine the precise range of acid and base stability of polymer complexes. Indeed, different polyzwitterions can produce phase behaviors that vary around different pH values ​​relative to the phase behavior observed for the pMPC-pAA system. Furthermore, it is possible to engineer phase behaviors that are opposite to those observed here, namely, base stability and acid stability. The feasibility of engineering this phenomenon using complementary synthetic approaches is conceivable.

[0127] The aspects of phase behavior presented here could have implications for the coordination of other polyzwitterionic materials. Designing similar synthetic polyzwitterions tailored to specific pH response criteria could be a fruitful avenue of research. The chemical composition could be adjusted to suit the stability or instability requirements at specific pH values. Furthermore, if such polyzwitterions were paired with polycations, then swapping the two charged groups on the polyzwitterionic monomers so that the permanently charged functional group is negative and the variably charged functional group is positive could produce the opposite phase behavior to that described in this work. These findings also apply to naturally occurring polyzwitterionic materials, namely proteins. The design of conjugates or adjuvants that interact with proteins to promote self-assembled structures could take into account the phenomena and concepts described and outlined here.

[0128] In a more general application, the pH-dependent phenomena described herein can be adapted to existing drug transport and delivery modalities. Furthermore, in the relatively chaotic environment of the GI tract, the influence of other factors, such as other ligands and physiological parameters other than pH and temperature (e.g., ionic strength), should also be considered. In addition to other problems that this teaching and pZC can address, the systems and methods of the present disclosure can also lay the foundation for addressing pressing issues in the pharmacology of GI drug delivery.

[0129] D. Package Model Load

[0130] These experiments demonstrate the encapsulation of a model load, BSA, in pZC droplets. First, the absorbance spectrum of fluorescently labeled BSA was measured to determine its peak absorption wavelength (495 nm). The fluorophore was selected so that its absorption peak was sufficiently far from the wavelength (550 nm) at which turbidity measurements were collected. Next, the three-component system was mixed to produce a solution containing protein and pZC droplets. Encouragingly, in the presence of BSA, the absorbance of the bulk solution followed the same pH-dependent behavior observed in the absence of BSA. However, this measurement is not sufficient to locate BSA inside or outside (or both) the pZC droplets. In order to test whether the protein separates in solution / where the protein separates, the sample was decelerated in a centrifuge to induce the bulk phase to separate into a polymer-rich lower phase and a polymer-poor supernatant. Since it is difficult to accurately measure the turbidity of a viscous, small-volume lower phase experimentally, absorbance measurements can be performed on the upper phase. At pH 2, when the complex is formed, the turbidity of the upper phase at 550 nm (corresponding to the polymer droplets) and the absorbance at 495 nm can be at their minimum baseline values, indicating that neither the protein nor the complex is present. This finding may indicate that at low pH, the protein colocalizes with the polyzwitterion complex. However, when the pH is increased to 4 and 5, the absorbance of the upper phase at 495 nm can reach a level corresponding to the absorbance of the bulk solution of the protein (about 0.2 au), which is consistent with the above results. Figure 11 This finding suggests that when the pH is increased, the protein is released and therefore cannot be separated from the polyzwitterion complex. Figure 12 As shown, the absorbance of the bulk solution of BSA-loaded pZC is highest at low pH. Figure 13 Shown is the absorbance of the upper phase after centrifugation as a function of pH.

[0131] Finally, to directly test the preferential separation of BSA from the polyzwitterionic complexes, fluorescence microscopy can be performed to visualize the colocalization of protein and chains into droplets. Figure 14 Representative images are shown.

[0132] E. NMR studies

[0133] After preparing a representative solution (at a peak pMPC:pAA mixing ratio of 30:70 at pH 2), the polymer-rich phase can be spun in a centrifuge and the polymer-poor supernatant removed. After lyophilizing the solvent from the remaining sample, solid-state NMR measurements were performed to probe the presence of each polymer. As expected, evidence of both pMPC and pAA can be observed in the resulting spectrum, indicating that both chains are involved in the coordination process. 10% D2O was added to the solid to improve spectral resolution. Cross-polarization (CP) NMR was obtained. Figure 15 The spectrum in 1HNMR obtained Figure 16 The spectrum in .

[0134] Incorporated by Reference

[0135] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, the specification shall prevail. In addition, any specific embodiment of the present disclosure that is a prior art may be expressly excluded from any one or more claims. Because such embodiments are deemed to be known to those of ordinary skill in the art, they may be excluded even if not expressly excluded herein. Any specific embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0136] Equivalence

[0137] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present disclosure. Therefore, the foregoing embodiments should be considered in all respects as illustrative and not limiting of the disclosure described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than the foregoing description, and all changes within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A polymer system, wherein the polymer system comprises a polyelectrolyte and a polyzwitterion, whereby the polyelectrolyte and the polyzwitterion form a polyzwitterionic complex.

2. The polymer system of claim 1, wherein the polyzwitterionic complex encapsulates a load.

3. The polymer system of claim 2, wherein the cargo is a therapeutic drug product.

4. The polymer system of claim 3, wherein the therapeutic drug product is selected from the group consisting of diabetes drugs, insulin, glucagon-like peptide-1 receptor agonists, gastric inhibitory peptides, probiotics, non-steroidal anti-inflammatory drugs, vaccines, monoclonal antibodies and nucleic acids.

5. The polymer system of any one of claims 1 to 4, wherein the polyelectrolyte is a pharmaceutically acceptable polyelectrolyte and the polyzwitterion is a pharmaceutically acceptable polyzwitterion.

6. The polymer system of claim 1, wherein the polyzwitterion is grafted onto a substrate.

7. The polymer system of any one of claims 1 to 6, wherein at least one of the polyelectrolyte and the polyzwitterion is a copolymer.

8. The polymer system of any one of claims 1 to 6, wherein the polyelectrolyte is selected from the group consisting of poly(acrylic acid), poly(styrene sulfonic acid), poly(methyl methacrylate), poly(dimethylaminoethyl methacrylate), poly(glutamic acid), poly(aspartic acid), heparan sulfate, hyaluronic acid, and copolymers thereof.

9. The polymer system of any one of claims 1 to 8, wherein the polyzwitterion is selected from the group of polyzwitterions consisting of phosphorylcholines, sulfobetaines, carboxybetaines, ammonium sulfonates, thiopyridinium betaines, cysteine ​​and copolymers thereof.

10. The polymer system of any one of claims 1-8, wherein the polyzwitterion is poly(2-methacryloyloxyethylphosphorylcholine).

11. The polymer system of any one of claims 1 to 10, wherein the polymer system further comprises a solvent.

12. The polymer system of claim 11, wherein the solvent comprises water, methanol, ethanol, ethylene glycol, propylene glycol, polyethylene glycol, or a combination thereof.

13. The polymer system of claim 11 or 12, wherein the solvent comprises one or more of sodium chloride, potassium chloride, sugar, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid and acetic acid.

14. The polymer system of any one of claims 1 to 10, wherein the polymer system is in a solid state.

15. A reconstituted polymer system comprising the polymer system of claim 14 and water.

16. The polymer system of any one of claims 1-13 or the restructured polymer system of claim 15, wherein the polyzwitterionic complex is formed at a pH of about 1 to about 3.

17. A polymer system comprising coacervates or precipitates formed by dipole-charge interactions or dipole-dipole interactions.

18. The polymer system of claim 17, comprising a polyelectrolyte and a polyzwitterion.

19. A method for delivering a cargo using a polymer system, wherein the polymer system comprises a polyzwitterion complex formed of a polyelectrolyte and a polyzwitterion and encapsulates the cargo, whereby when the pH of the surrounding environment changes, the polyzwitterion complex is unwound to release the encapsulated cargo.

20. The method of claim 19, wherein the pH of the surrounding environment is increased to release the encapsulated load.

21. The polymer system of claim 19 or 20, wherein the encapsulated cargo is a therapeutic drug product.

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