A coacervate and its preparation method and application

Abstract

The present application relates to a kind of condensate and its preparation method and application.The condensate is formed by the self-assembly of hydrophilic polymer chain with hydrophobic group modified end group, the hydrophilic polymer chain is selected from polyethylene glycol and its block copolymer, polypropylene glycol and its block copolymer, polyacrylic acid and its block copolymer, polyamino acid and its block copolymer, the hydrophobic group is selected from alkane group, aromatic hydrophobic group or aripiprazole, etoposide, methyltestosterone, triamcinolone acetonide, dexamethasone acetate, fluocinolone acetonide and other hydrophobic compounds.The condensate of the present application is simple to prepare, non-toxic to physiology, as prodrug or drug delivery system, can play the function of long-acting drug release, long in vivo retention time and has lubricating effect.

Description

Technical Field

[0001] The technical solution of this invention belongs to the field of biomedicine, specifically, it relates to an aggregate and its preparation method, as well as the application of the aggregate in the preparation of drug precursors or drug delivery. Background Technology

[0002] Polyethylene glycol (PEG) retains the good solubility of polyethylene glycol in water and can also impart flexibility, anticoagulation, and anti-macrophage phagocytosis effects to modified biomolecules. PEG has applications in many fields, including medical research, drug release, and nanotechnology. Utilizing the hydrophilicity of polyethylene glycol, there have been reports of drugs being conjugated to it to improve water solubility. CN107137715A discloses a PEG-conjugated prodrug of paliperidone, which uses amino acids or oligopeptides composed of amino acids as linkers to modify paliperidone through PEGylation, thereby extending the drug's half-life and increasing its water solubility. There are also studies (Sun Zhouliang et al., Strait Pharmaceutical Journal, 2008, 20(12):7-10) that have combined curcumin with polyethylene glycol (PEG) to overcome the problems of poor water solubility and low bioavailability of curcumin. Enzon's PEGylated camptothecin utilizes one molecule of PEG to conjugate two drug molecules, which can increase the drug loading; at the same time, PEG modification also increases the water solubility of small molecule drugs.

[0003] However, existing drug formulations still suffer from problems such as easy dilution by body fluids, low absorption efficiency, and short residence time. Therefore, the development of novel delivery formulations remains a research hotspot. Controlled-release drug delivery has steadily progressed in medical practice. To date, at least 63 long-acting drug products have received FDA approval, including long-acting contraceptives, prolonged hormone suppression, opioid and alcohol addiction treatments, and topical ophthalmic medications, providing drugs for periods ranging from one month to six years or even longer. However, long-acting drug delivery formulations still face several technical challenges: firstly, determining the dosage and release duration, which is related to drug efficacy and injection volume limitations; secondly, managing the physicochemical stability of proteins and other biological products during microencapsulation and sustained-release processes. Furthermore, systemic and topical drug delivery (such as ocular and joint delivery), as well as immunotherapies, are also promising research directions for long-acting drug delivery formulations.

[0004] As an emerging biomedical material in recent years, condensates have been extensively studied in fields such as biointerface adhesion, macromolecular drug carriers, liquid robots, and artificial cells and membraneless organelles. Based on the classic Spacer-Sticker theory of condensed droplet construction, the Spacer provides structural support or background stability within a molecular or molecular region, while the Stick, through supramolecular interactions, guides the attraction or binding of biomolecules, enabling self-assembly of condensate phases through liquid-liquid phase separation. Although condensate materials have been explored in drug delivery, the development of condensate phases into long-acting drug formulations remains an area for further research. Summary of the Invention

[0005] One of the objectives of this application is to provide a novel aggregate formed by the self-assembly of hydrophilic polymer chains with hydrophobic end groups.

[0006] In some embodiments, the hydrophilic polymer chain is selected from one or more of polyethylene glycol and its block copolymers, polypropylene glycol and its block copolymers, polyacrylic acid and its block copolymers, and polyamino acids and their block copolymers.

[0007] In some embodiments, the hydrophilic polymer chain is selected from polyethylene glycol, polypropylene glycol, polyacrylic acid, polyleucine, polyglutamic acid, polyaspartic acid, polylysine, etc. In some embodiments, the hydrophilic polymer chain is selected from block copolymers containing polyethylene glycol, polypropylene glycol, polyacrylic acid, polyleucine, polyglutamic acid, polyaspartic acid, or polylysine.

[0008] In some embodiments, the molecular weight of the polymer chain is 200-1200, and in some embodiments the molecular weight of the polymer chain can be 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or 1200.

[0009] In some embodiments, the hydrophobic group is selected from alkane groups, aromatic hydrophobic groups, or hydrophobic compounds such as aripiprazole, etoposide, methyltestosterone, triamcinolone acetonide, dexamethasone acetate, and fluocinolone acetonide. In some embodiments, the alkane group has 2-50 carbon atoms; the aromatic hydrophobic group is selected from phenylboronic acid esters and their derivatives, aromatic hydrocarbons and their derivatives, and azobenzene and its derivatives; in some embodiments, the aromatic hydrophobic group can be benzene and its derivatives, naphthalene and its derivatives; in some embodiments, the aromatic hydrophobic compound can be selected from benzene, toluene, ethylbenzene, propylbenzene, cumene, butylbenzene, tert-butylbenzene, alkylbenzene, xylene, naphthalene, phenanthrene, diphenylmethane, biphenyl, azobenzene and its derivatives, etc.

[0010] In some embodiments, the hydrophobic groups at both ends of the modified hydrophilic polymer chain are different hydrophobic groups. For example, in some embodiments, one end of the hydrophilic polymer chain is modified with phenylboronic acid esters and their derivatives, and the other end is modified with alkane groups, other aromatic hydrophobic groups, or hydrophobic compounds such as aripiprazole, etoposide, methyltestosterone, triamcinolone, dexamethasone acetate, and fluocinolone acetonide.

[0011] In some embodiments, the hydrophobic group is linked to the hydrophilic polymer chain via an ester bond, amide bond, ether bond, or urea bond. In some embodiments, the degree of polymerization of the hydrophilic polymer chain is 3-20.

[0012] Another objective of this application is to provide a method for preparing aggregates, comprising the following steps: adding hydrophilic polymer chains with hydrophobic end groups to water, physiological solutions, or other aqueous phases, and self-assembling to form aggregates. Wherein, the degree of polymerization n of the hydrophilic polymer chains is 3-20; the hydrophobic groups are selected from alkane groups, aromatic hydrophobic groups, or hydrophobic compounds such as aripiprazole, etoposide, methyltestosterone, triamcinolone, dexamethasone acetate, and fluocinolone acetonide.

[0013] In some embodiments, the other aqueous phase includes, but is not limited to, cell culture medium, tissue culture medium, and body fluid.

[0014] In some embodiments, the method for preparing the hydrophilic polymer chain with the hydrophobic group-modified end group includes the following steps: subjecting the hydrophilic polymer chain or its end-group-modified derivative to an esterification, amidation, ureation, or Williamson reaction with a compound containing the hydrophobic group.

[0015] Another object of this application is to provide the use of the said aggregate in the preparation of precursor drugs and lubricants.

[0016] The aggregates of this application, formed by the self-assembly of therapeutically active compounds after modification of the end groups of the hydrophilic polymer chains, can serve as prodrugs. In vivo, they release the active compounds through simple hydrolysis to exert pharmacological effects. The aggregates of this application are simple to prepare, have no physiological toxicity, and, as drug prodrugs, provide long-lasting drug release. Furthermore, these aggregates possess the following characteristics: first, they are immiscible with the aqueous phase, resulting in a long retention time as a drug formulation; second, they have a certain lubricating effect, which is advantageous for drug delivery, for example, at joint sites.

[0017] Another object of this application is to provide the application of the said condensate in drug delivery. The condensate of this application, as a delivery system, has a high drug content and can target and release drugs in vivo by attaching pH- and reactive oxygen species-responsive groups to one or both ends of a hydrophilic polymer chain segment. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the preparation of self-assembled aggregates of hydrophilic polymer chains with hydrophobic group-modified end groups.

[0019] Figure 2 Physical images and optical microscope images of the liquid-liquid phase separation of polyethylene glycol butyrate condensates (a), flow properties shown (b).

[0020] Figure 3 Images (a) and rheological tests (b) of butyric acid aggregates after incubation in different simulated solutions for 48 h.

[0021] Figure 4 Cumulative release-time fitting curves (Q%-t) of butyrate aggregates in SGF(a), SIF(b), and PBS(c).

[0022] Figure 5 Figure 5-1 : Flowchart of mouse modeling and treatment (a); Weight change of mice in different treatment groups over 7 days (b); DAI disease activity index (c); Figure 5-2 Images of colon tissue (d) and colon length (e); representative H&E and alicin blue staining images of mouse colon tissue; representative fluorescence images of ZO-1 and Occludin-1 protein expression (f); histopathological score (g); semi-quantitative fluorescence analysis results of ZO-1 (h) and Occludin-1 (i).

[0023] Figure 6 Figure 1: Mouse body weight change (a); mouse body weight change over 10 weeks (b); mouse food intake (c); mouse serum levels of TG, TC, HDL, and LDL (d); whole-body photograph and CT scan of mice (e); mouse eWAT, iWAT, and BAT weights (f).

[0024] Figure 7 Acid resistance test (a), alkali resistance test (c), and corresponding turbidity statistics (b, d) of polyethylene glycol ibuprofen aggregates.

[0025] Figure 8 Drug release curves of polyethylene glycol ibuprofen aggregates over 14 days.

[0026] Figure 9 Schematic diagram (a) and dynamic friction coefficient (b) of polyethylene glycol ibuprofen aggregate in vitro cartilage friction device.

[0027] Figure 10 Imaging of a joint-retention animal containing polyethylene glycol ibuprofen aggregates.

[0028] Figure 11A schematic diagram (a) of adding hydrogen peroxide to polyethylene glycol phenylboronic acid ester aggregates and the turbidity of the solution at different hydrogen peroxide concentrations (b).

[0029] Figure 12 Optical microscopic images of polyethylene glycol condensates with different end groups, (a) fluocinolone acetonide,

[0030] (b) Triamcinolone acetonide, (c) Dexamethasone acetate, (d) Etogestene.

[0031] Figure 13 (a) 1H NMR spectrum of estradiol-modified polyethylene glycol; (b) Optical micrograph of estradiol-modified polyethylene glycol in water.

[0032] Figure 14 Optical microscopic images of polyethylene glycol condensates with different end groups, (a) aripiprazole,

[0033] (b) Butyl isocyanate, (c) Hexyl isocyanate.

[0034] Figure 15 (a) Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to analyze the molecular weight change of PEG before and after the modification patch. (b) Macroscopic images of aggregates formed by P600B2, P800B2 and P1000B2.

[0035] Figure 16 The formation of aggregates from monodisperse polyethylene glycol molecules with different end groups.

[0036] Figure 17 (a) PPGA-But NMR 1H spectrum; (b) PPGA-Hex NMR 1H spectrum.

[0037] Figure 18 (a) Physical image of PPG condensates undergoing liquid-liquid phase separation; (b) Fluorescence recovery image of PPG condensate droplets after photobleaching. Detailed Implementation

[0038] Unless otherwise stated, all reagents used in the embodiments of this application are commercially available products.

[0039] The design of the self-assembled condensate in this application utilizes a spacer-sticker model constructed through various supramolecular interactions, such as charge interactions and hydrophobic effects. Considering that most drugs are hydrophobic, hydrophilic polymer segments can be selected as spacers, with hydrophobic drugs covalently linked at both ends via ester bonds or other bonds to act as stickers. The condensate then self-assembles through hydrophobic interactions to form a condensate. Figure 1 ).

[0040] Under the action of an alkaline catalyst (including but not limited to dibutyltin dilaurate, triethylamine, or dicyclohexyldiimide), a Spacer compound (polyethylene glycol or its derivative) and a Sticker compound (hydrophobic compound) are added to a three-necked flask at a molar ratio of (1:2) to (1:4), and esterification, amidation, ureation, or Williamson reaction is carried out at room temperature to obtain a mixture. The mixture is purified by silica gel column chromatography to obtain agglomerate precursor compounds. Adding the agglomerate precursor compounds to water, physiological solutions, or other aqueous phases allows for self-assembly to form agglomerates, resulting in liquid-liquid phase separation. The degree of polymerization (n) of the hydrophilic polymer segments and their derivatives is 3-20; the connecting groups at both ends can be selected from alkane compounds, aromatic compounds, or other hydrophobic compounds, as shown in some examples below.

[0041]

[0042] In some embodiments, under the action of an alkaline catalyst, hydrophilic polymer segments or their derivatives are added to a three-necked flask with an acyl halide containing hydrocarbon or aromatic hydrophobic groups, or dexamethasone acetate, at a molar ratio of (1:2) to (1:4), and esterification or amidation reactions are carried out at room temperature to obtain a mixture. The mixture is purified to obtain a hydrophilic polymer chain modified with hydrocarbon, aromatic, or other hydrophobic groups at both ends. In some embodiments, bromomethylphenylboronic acid ester is reacted with a hydrophilic polymer chain or its derivative containing alcohol groups at the ends to obtain a hydrophilic polymer chain with phenylboronic acid esters attached at both ends. In some embodiments, a hydrophilic polymer chain or its derivative containing carboxyl groups at the ends is added to a three-necked flask with methyltestosterone, etoposide, fluocinolone acetonide, triamcinolone acetonide, aripiprazole, etc., at a molar ratio of (1:2) to (1:4), and esterification or amidation reactions are carried out at room temperature to obtain a mixture. The mixture is purified to obtain a hydrophilic polymer chain with the hydrophobic groups attached at both ends. In some embodiments, the hydrophilic polymer chain segment with aminated terminal groups undergoes a ureation reaction with isocyanate in a molar ratio of (1:2) to (1:4) to obtain a hydrophilic polymer chain with hydrophobic group-modified terminal groups.

[0043] Example 1. Preparation of self-assembled polyethylene glycol butyrate aggregates

[0044] Under the action of an alkaline catalyst (triethylamine), 10 g of polyethylene glycol (molecular weight 300) and butyryl chloride were added to a three-necked flask at a molar ratio of 1:2. An appropriate amount of ultra-dry dichloromethane was then added, and the mixture was stirred thoroughly. Esterification was carried out at room temperature for 24 hours to obtain a mixture. The mixture was purified by silica gel column chromatography using a mobile phase of dichloromethane and methanol in a ratio of 90:10 to 80:20 (v / v). The solution collected from column volumes 3 to 8 was a polyethylene glycol butyric acid molecular solution, which was then purified by vacuum distillation to obtain polyethylene glycol butyric acid. An appropriate amount of polyethylene glycol butyric acid was added to water, resulting in liquid-liquid phase separation to obtain polyethylene glycol butyric acid aggregates.

[0045] Example 2. Characterization of polyethylene glycol butyrate aggregates

[0046] An appropriate amount of polyethylene glycol butyric acid condensate and deionized water were added sequentially to a glass bottle, and the mixture was vortexed for 10 seconds to ensure homogeneity, resulting in a suspension of condensed droplets. The morphology of the condensed droplets was characterized using an optical microscope.

[0047] The butyric acid aggregates are immiscible with water (e.g. Figure 2 (As shown in a). An optical microscope was used to image a 50 mg / ml butyric acid aggregate suspension, and uniformly distributed spherical aggregate droplets with a particle size range of 50-70 μm were observed within the field of view. Figure 2 a). The butyric acid aggregates can flow out of the tilted glass sample vial, indicating that the butyric acid aggregates have macroscopic fluidity ( Figure 2 b) It is suitable for various administration methods such as oral, injection, rectal irrigation, eye drops, nasal spray, and oral spray (inhaler).

[0048] Example 3. Stability test of polyethylene glycol butyrate aggregates

[0049] During oral administration, traditional complex aggregates are susceptible to depolymerization into single-phase solutions due to the harsh environment of the gastrointestinal tract (e.g., pH ranges from 1-2.5 in the stomach to 6.15-7.88 in the intestine; and extensive salt concentration variations). Therefore, to investigate the in vivo stability of polyethylene glycol butyrate aggregates, they were immersed in simulated gastric juice (SGF), simulated intestinal juice (SIF), and buffer solutions containing 0.15, 0.25, 0.5 M, and 1 M salt concentrations, respectively. The stability was assessed by measuring changes in the macroscopic rheological properties of the aggregates.

[0050] After incubation at 37°C in different simulated gastrointestinal tract solutions for 48 hours, it was observed that the polyethylene glycol butyrate aggregates did not depolymerize and remained separated. Figure 3 a). Tests showed that the loss modulus (G”) of the polyethylene glycol butyrate aggregate was greater than its storage modulus (G’), and the change was not significant. Figure 3b) indicates that after oral administration, the polyethylene glycol butyrate aggregates maintain an immiscible liquid state and remain stable in the harsh environment of the gastrointestinal tract. To further determine the stability of the aggregates under physiological conditions, their rheological properties were investigated under different salt concentrations. The results showed that the polyethylene glycol butyrate aggregates can undergo liquid-liquid phase separation to form a condensed phase in environments with a wide range of salt concentrations, and their loss modulus and storage modulus remain stable without significant changes (see [link to relevant documentation]). Figure 3 a,c).

[0051] Example 4. Butyric acid release performance of polyethylene glycol butyric acid aggregates

[0052] To investigate the butyrate aggregate's effect as a prodrug in the sustained-release of butyrate, equal amounts of polyethylene glycol butyrate aggregate were added to 2 ml of simulated gastric juice (SGF), simulated intestinal juice (SIF), and PBS (pH 6.8), respectively, and incubated at 37°C. Supernatants were collected at corresponding time points. A suitable amount of the supernatant was then subjected to HPLC analysis to determine the butyrate release amount (Q). ％ The formula is as follows.

[0053]

[0054] In the formula, Ct represents the concentration of butyric acid in the release medium at time t (mg / ml), Ci represents the concentration of butyric acid in the release medium at time t-1 (mg / ml), Vi represents the sampling volume at time t-1 (ml), and W represents the amount of butyric acid added (mg).

[0055] The sample content was determined by HPLC, and the method is as follows: chromatographic column: PotenSil C 18 (4.6×250mm, 5μm), mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution = 20:80 (v / v); flow rate: 1ml / min; column temperature: 30℃; injection volume: 10μl; detection wavelength: 210nm.

[0056] This embodiment uses glyceryl tributyrate, which is also linked to butyric acid via ester bonds, as a control to investigate the in vitro release of polyethylene glycol butyric acid aggregates. Figure 4 As shown in Figure a, the release of butyrate aggregates in the SGF simulated solution was less than 2% after 10 hours, and was slowly released over 3 days, indicating that butyrate aggregates are stable in the acidic environment of the stomach. However, in the SIF simulated solution, butyrate aggregates released a large amount of butyrate within 30 minutes, with a release rate 3.79 times that of tributyrate glyceride, and a release rate of 4.42 times after 2 hours. Figure 4 b). Compared with PBS without added pancreatic lipase, both butyrate prodrugs showed butyrate release of less than 1% within 12 hours, with no significant butyrate release behavior. Figure 4c). In vitro simulated drug release experiments showed that, under alkaline intestinal conditions, butyrate aggregates undergo slow hydrolysis of ester bonds due to the action of pancreatic lipase, releasing sufficient butyrate to exert its effect.

[0057] Example 5. Therapeutic effect of polyethylene glycol butyrate aggregates on colitis in mice.

[0058] This study investigated the therapeutic effect of polyethylene glycol butyrate aggregates on colitis in mice. A mouse colitis model was established using 3% sodium dextran sulfate (DSS, 4 kDa). Female C57BL / 6J mice were acclimatized for one week, with normal access to water and food. The mice were then given free access to 3% DSS. Successful establishment of the colitis model was indicated by observed lethargy, significantly reduced activity, rapid weight loss, and unformed or even bloody stools. Mice were randomly divided into 7 groups (n=6): a healthy group (PBS); a modeling group (3% DSS); a 150 mg / kg butyrate aggregate group (Peg-But, 3% DSS); a 150 mg / kg tributyrate group (Tributyri, 3% DSS); and a 150 mg / kg sodium butyrate group (But, 3% DSS). During the modeling process, mice were administered the same dose of butyrate daily via oral gavage, as illustrated in Figure 5a. Among them, the healthy group, the tributyrate group, and the sodium butyrate group served as the control group.

[0059] Throughout the experiment, mouse body weight, fecal viscosity, and fecal occult blood were recorded daily. Curves showing changes in mouse body weight and DAI (Disease Activity Index) over time were plotted to evaluate the therapeutic effects of different butyrate aggregates. At the end of treatment on day 9, mice were euthanized, and intact intestinal tissue was harvested. Colon length was measured and photographed. Colon tissue samples were obtained from dissected mice. Intestinal contents were washed with physiological saline, and the samples were fixed in 4% paraformaldehyde solution (for 48 hours) and embedded in paraffin. Paraffin-embedded colon tissue sections (4 μm) were observed using H&E and Alcian blue staining. Furthermore, sections were stained with AlexaFluor-488-conjugated anti-ZO-1 antibody and AlexaFluor-488-conjugated anti-Occludin-1 antibody, respectively, and DAPI was used to stain cell nuclei.

[0060] The daily body weight and DAI score of mice in each group are shown in Figure 5. Mice in the normal group had normal activity and diet, stable body weight, and minimal changes in DAI score (Figure 5b). With increasing treatment duration, the rate of body weight loss in the butyrate aggregate group slowed to varying degrees; while the body weight of mice in the DSS group, tributyrate group, and sodium butyrate group decreased significantly (p < 0.001, p < 0.05, p < 0.001). On day 7, compared with the model group, the body weight of mice in the butyrate aggregate group was significantly increased (p < 0.05), and the DAI score was significantly decreased (p < 0.001); compared with the sodium butyrate group, the body weight and DAI score of mice in the butyrate aggregate group were significantly increased (p < 0.05, p < 0.001). This indicates that butyrate aggregates can effectively restore the body weight of mice with colitis, reduce intestinal inflammation, and have a good therapeutic effect.

[0061] The colon length of mice in the normal group was 7.95±0.46 cm, while that in the model group was 6.12±0.62 cm, indicating damage to the colonic tissue (Figure 5d), resulting in a significantly shorter colon length compared to the normal group (p<0.00001). However, after intervention with butyrate aggregates, the colon length of mice significantly increased compared to the model group (Figure 5e, p<0.00001). Furthermore, the butyrate aggregate treatment group showed significant differences in its effect on colon length compared to the tributyrate group and the sodium butyrate group (p<0.01, p<0.00001). This indicates that butyrate aggregate treatment can effectively restore colon length in colitis mice and has a better therapeutic effect.

[0062] As shown in Figure 5f, the histopathological sections of the colon tissue of healthy mice showed intact colonic structure and mucosal epithelium, uniform crypt distribution, and no obvious necrosis. In contrast, the colon of mice in the model group showed mucosal damage and edema, crypt branching, reduced goblet cells, and inflammatory cell infiltration. In colitis mice treated with sodium butyrate, the number of goblet cells in the colonic tissue was significantly reduced, and large mucus vacuoles were frequently observed, indicating that sodium butyrate was ineffective at this dose. Compared to the model group, the colonic mucosal epithelial damage in the butyrate aggregate group and the tricresyl triglyceride group was restored, the colonic cell morphology was relatively regular, and inflammatory cell infiltration was reduced. Based on the histopathological scoring of tissue damage (Figure 5g), the histopathological score of the colonic tissue in the butyrate aggregate group was significantly lower than that in the DSS group and the sodium butyrate group (p<0.01, p<0.05), indicating that the butyrate aggregate treatment in this embodiment can effectively improve tissue damage in colitis mice and has a good therapeutic effect.

[0063] Intestinal mucin, synthesized and secreted by goblet cells in the colon, is the first line of defense against harmful substances entering the body and is closely related to maintaining intestinal homeostasis. As shown in Figure 5f, mucin is evenly distributed in the colonic tissue of healthy mice, and a complete mucus layer can be observed. In mice with colitis induced by DSS, tissue damage is severe, goblet cells secreting mucin are almost entirely absent, and a complete mucus layer cannot be observed. In mice in the tributyrate and sodium butyrate groups, mucin expression is increased in the colonic tissue, but the distribution is uneven. In contrast, the butyrate aggregate group of mice has a large number of goblet cells in the crypts of the colonic tissue, and mucin expression is significantly restored, similar to the healthy group, with a relatively complete mucus layer still observable. These results indicate that butyrate aggregate intervention can effectively improve the reduction of intestinal goblet cells under inflammatory conditions, maintain the integrity of the mucus layer, and alleviate intestinal inflammation.

[0064] ZO-1 and Occludin-1 proteins are tight junction-related proteins in the gut and play a crucial role in maintaining intestinal homeostasis. As shown in Figure 5f, almost no expression of ZO-1 and Occludin-1 proteins was observed in the DSS group, while the expression levels of ZO-1 and Occludin-1 proteins in the colonic tissue of colitis mice treated with butyrate condensates were significantly increased, neatly arranged in the intercellular spaces, and showed no significant difference from the expression in healthy mice. Small amounts of ZO-1 and Occludin-1 protein expression were observed in the tributyrate group and sodium butyrate group, showing a discontinuous distribution. As shown in Figure 5h,i, compared with the butyrate condensate group, the expression of ZO-1 protein in the tributyrate group and sodium butyrate group was significantly decreased (p < 0.001, p < 0.00001). Meanwhile, the fluorescence expression of Occludin-1 protein in the sodium butyrate group also decreased significantly (p < 0.001), indicating that at this dose, the therapeutic effect of clinical drugs (sodium butyrate and tricresyl triglyceride) was not as good as that of butyrate aggregates. This suggests that butyrate aggregates can significantly increase the expression of tightness-related proteins in the intestinal tissue of colitis mice, and have a role in repairing the intestinal barrier.

[0065] Example 6. Therapeutic effect of polyethylene glycol butyrate aggregates on obese mice

[0066] Butyrate, a short-chain fatty acid, is the final product of microbial fermentation of dietary fiber in the intestinal lumen. Studies have shown that butyrate not only plays an important role in maintaining intestinal homeostasis but can also enter the bloodstream to directly affect the function of extraintestinal tissues. Butyrate can improve obesity and glucose metabolism by promoting intestinal gluconeogenesis (IGN), specifically through the cyclic adenosine monophosphate (cAMP) pathway. Butyrate can mediate the upregulation of calcium or calmodulin-dependent protein kinase II expression in hepatocytes by G protein-coupled receptors 41 and 43, and inhibit histone deacetylase 1 expression in Hep1-6 cells, thereby improving hepatic steatosis, abnormal lipid metabolism, and delaying the onset of obesity. In addition, butyrate can also inhibit appetite by promoting the release of anorexia nervosa hormones in the gut and reducing the activity of neuropeptide Y-mediated appetite neurons, the nucleus tractus solitarius in the brainstem, and the dorsal vagal complex; however, this inhibitory effect may be unrelated to fatty acid receptors. Promoting thermogenesis in adipose tissue to increase energy expenditure is also a mechanism by which butyrate inhibits obesity. Under the action of monocarboxylic acid transporter 1 (MCT1) and acyl-CoA medium chain synthase 3 (ACSM3), butyrate is transported into adipocytes and promotes thermogenesis in adipocytes by activating intracellular lysine-specific demethylase (LSD1).

[0067] To investigate the therapeutic effect of butyrate aggregates on obese mice, an obesity model was established by feeding mice with a high-fat diet (60% energy supplied by fat) for 10 weeks, with a control group consisting of mice fed a normal diet (10% energy supplied by fat). Male C57BL / 6J mice were acclimatized for one week, during which time they were provided with normal water and food. Mice were randomly divided into 7 groups (n=5): a healthy group (PBS); a model group (HFD); a 150 mg / kg butyrate aggregate group (Peg-But, HFD); and a 600 mg / kg sodium butyrate group (Butyrate, HFDS). During the modeling process, mice were administered the same dose of butyrate daily via oral gavage, as illustrated in the diagram below. The healthy group and the sodium butyrate group served as control groups.

[0068] During the 10-week animal experiment, the mice's food intake was recorded daily, and their weight was recorded weekly. At week 9, a whole-body CT scan was performed on the mice. After the experiment, the mice were euthanized, and whole blood was collected for serum separation and quantitative analysis of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Additionally, epididymal fat (eWAT), inguinal fat (iWAT), and brown adipose tissue (BAT) were collected and weighed.

[0069] First, assuming no significant difference in initial body weight among the groups, the inventors evaluated the effects of butyrate aggregates and sodium butyrate on body weight in mice on a high-fat diet. At week 10, the average body weight of the healthy group mice was 33.80 g, while the average body weight of the model group mice (57.18 g) increased by 69.17% compared to the control group. Figure 6 (a, b) Under the intervention of butyrate agglomerates or sodium butyrate, the body weight of each group decreased by 80.77% and 27.63% compared with the model group, respectively. Furthermore, the food intake and energy intake of mice in the butyrate agglomerate or sodium butyrate intervention groups were not different from those in the model group, indicating that butyrate agglomerates do not inhibit body weight gain by affecting food intake in mice. Figure 6 c). Weight is not the only indicator of obesity; obese individuals often have dyslipidemia, indicating a metabolic disorder. Therefore, the effects of butyrate agglutinates or sodium butyrate on dyslipidemia under a high-fat diet were assessed by measuring serum concentrations of TG, TC, HDL, and LDL. Butyrate agglutinates significantly reduced serum levels of TG, TC, HDL, and LDL, indicating that butyrate agglutinates improved high-fat diet-induced dyslipidemia. Figure 6 d). However, sodium butyrate intervention did not significantly improve dyslipidemia. Whole-body CT scans of mice at week 9 revealed that butyrate aggregates effectively reduced the accumulation of subcutaneous and visceral fat, achieving weight loss. Figure 6 e). Combining Figure 6 As shown in f, the eWAT, iWAT, and BAT weights of the model group mice were significantly higher than those of the control group, indicating that a high-fat diet promotes lipid accumulation in adipose tissue. However, butyrate aggregates significantly reduced the weights of eWAT, iWAT, and BAT. This suggests that butyrate aggregates can significantly inhibit adipose tissue expansion and reduce lipid accumulation.

[0070] Example 7. Preparation of polyethylene glycol ibuprofen aggregates

[0071] Under the action of an alkaline catalyst (triethylamine), polyethylene glycol (10 g) with molecular weights of 400 (n=8), 600, and 1000, respectively, was added to a three-necked flask with ibuprofen amide chloride at a molar ratio of 1:3. An appropriate amount of ultra-dry dichloromethane was then added, and the mixture was stirred thoroughly with a stir bar. Esterification was carried out at room temperature for 24 hours to obtain a mixture. The mixture was then passed through a C1 filter with a mobile phase of water and methanol in a ratio of 90:1 to 80:20 (v / v). 18 Purification was performed by column chromatography, and the solution from column volumes 10 to 13 was collected and subjected to vacuum distillation to obtain polyethylene glycol (PEG) aggregate monomers. An appropriate amount of PEG aggregate monomers was added to water, resulting in liquid-liquid phase separation to obtain ibuprofen prodrug aggregates.

[0072] Example 8. Stability test of prepared polyethylene glycol ibuprofen aggregates

[0073] The acid and alkali resistance of condensed liquid prodrugs refers to their ability to remain stable over a relatively wide physiological pH range for extended periods. Good acid and alkali resistance allows for a wider range of applications, such as addressing acidification caused by local inflammation. To make the tests more representative, an acidic environment was set at pH 3, and an alkaline environment at pH 11, and turbidity was statistically analyzed using software. Ibuprofen condensates prepared from polyethylene glycol with molecular weights of 400, 600, and 1000 were named IP400, IP600, and IP1000, respectively.

[0074] The results are as follows Figure 7 As shown, under the same conditions, the turbidity is higher under alkaline conditions and lower under acidic conditions, indicating that the condensed liquid prodrug is more stable under acidic conditions. This may be related to the fact that ester bonds are more easily hydrolyzed under alkaline conditions. Meanwhile, under the same conditions, the turbidity decreases with increasing chain length, indicating that its stability increases with chain length.

[0075] Example 9. In vitro release experiment of polyethylene glycol ibuprofen aggregates

[0076] Drug release from ibuprofen aggregates was simulated under physiological conditions in PBS (pH 7.4) at 37°C. Ibuprofen aggregates of different chain lengths were divided into three parallel samples, with each sample containing 0.15 mol of material. The corresponding mass values ​​for IP1000, IP600, and IP400 were 211.9, 151.9, and 121.9 mg, respectively. 1.5 mL of PBS solution was added to each sample. On days 1, 2, 3, 4, 5, 6, 7, and 14, 750 μL of the supernatant was collected after centrifugation, and the ibuprofen content was determined by HPLC. Simultaneously, 750 μL of fresh PBS solution was added.

[0077] The ibuprofen content in the supernatant was determined according to the detection method for ibuprofen in the 2020 edition of the Chinese Pharmacopoeia. The specific method is as follows: using C... 18 Quantitative analysis of in vitro degradation products was performed by HPLC using a PotenSil column (5 μm, 4.6 × 150 mm) on a Waters 2695 separation module equipped with a Waters 2489 dual absorbance detector. The system was connected to a Dell computer running Empower software. The mobile phase was sodium acetate buffer (pH 2.5)-acetonitrile (40:60, v / v); the detection wavelength was 263 nm; and the injection volume was 20 μL. Collected samples were first diluted with an equal volume of methanol and filtered through a 0.22 μm filter before detection.

[0078] The results are as follows Figure 8As shown, no burst release was observed in the degradation curves of the ibuprofen aggregates; the drug was released from its respective aggregate liquid in a controlled manner, and the 14-day release of ibuprofen from the aggregates increased from 3% to 8% of the loading as the chain length and molecular weight increased from 400 to 1000. This can be attributed to the higher solubility and higher water content of the longer-chain prodrug aggregates. In summary, the 14-day release of the prodrug aggregates can be adjusted between 3% and 8% of the loading, demonstrating its promising application as a long-acting sustained-release formulation.

[0079] Example 10. In vitro cartilage interface friction experiment of polyethylene glycol ibuprofen aggregates

[0080] Since the daily activities of the knee joint mainly involve knee extension and flexion along the horizontal axis and rotational movements around the lower leg axis, friction tests of the cartilage interface can be conducted using sliding and rotating devices based on these two activity patterns. Sliding devices are suitable for biomechanical studies modeling physiological kinematics, while rotating devices are suitable for evaluating lubrication at interfaces (such as articular cartilage interfaces). Based on existing research reports, this method uses a rotating device (rotational rheometer) for in vitro cartilage interface friction tests. Throughout the test, the cartilage core and cartilage ring samples are placed in parallel, axially compressed, and rotated relative to each other. The test setup is as follows: Figure 9 As shown in a.

[0081] Preparation before testing: (1) First, record the thickness of the cartilage samples using calipers, and the overall thickness of each cartilage sample is considered as the sum of the thickness of the cartilage core and the cartilage ring. (2) Next, fix the cartilage ring sample to an 8mm parallel plate clamp with cyanoacrylate glue, and fix the cartilage core sample to the center of the bottom of a 3.5cm culture dish, which is fixed on a plate at the bottom. (3) Then, completely immerse the cartilage core sample in PBS solution. (4) Before starting the test, take 100ul of agglomerated prodrug and place it on the surface of the cartilage core sample to simulate the biological lubricant injected into the joint cavity.

[0082] The testing process mainly consists of the following four stages: (1) In order to simulate the compression level of physiological kinematics, the cartilage sample was compressed to 82% of the original overall thickness of the cartilage sample, so that the upper and lower cartilage samples could fully contact each other. (2) Then, the sample was pre-sheared by rotating it twice in both the forward and reverse directions at an effective sliding speed of 0.3 mm / s. The pre-shearing stage was repeated twice to keep the surface of the cartilage sample flat. (3) In order to eliminate the fluid accumulation between the cartilages, a stress relaxation period of 3600 seconds was maintained to promote fluid decompression. (4) Considering that the friction coefficient of the joint during actual activity will be affected by long-term axial load (such as standing or sitting), the cartilage sample was kept in contact for a long time (1200 seconds) and a short time before rotation, respectively. Then, the sample was rotated twice in the forward direction and twice in the reverse direction at an effective sliding speed of 0.3 mm / s. During each rotation of the sample, the axial load (N) and torque (τ) were recorded, and the dynamic friction coefficient (μk) was calculated. The calculation formula is as follows:

[0083] μk=τ / (Reff×N)

[0084] During the testing process, in order to simulate the compression and rotation levels of physiological kinematics, the cartilage sample was compressed to 82% of the original overall sample thickness to ensure full contact between the upper and lower cartilage samples. Then, the dynamic friction coefficient of the cartilage interface was measured by rotating forward and backward at an effective sliding speed of 0.3 mm / s.

[0085] like Figure 9 As shown in b, under the same resting time of cartilage samples and the action of different biological lubricants, the dynamic friction coefficient of the coagulated prodrug was significantly lower than that of the PBS group for both normal and cartilage samples, showing a statistically significant difference. The coefficient was also similar to that of the HA group, indicating that the coagulated prodrug layer can effectively reduce the friction coefficient of normal cartilage under physiological joint load and possesses good lubrication performance. This may be because the polyethylene glycol chains in the coagulated prodrug can bind with water molecules to form a hydrated lubricating layer, which can play a lubricating role at the cartilage interface. Secondly, the lubrication performance of the coagulated prodrug IP-1000 is better than that of IP-400. This may be attributed to the shorter polyethylene glycol chains in the IP-400 coagulated prodrug, which results in stronger interaction forces at its two ends of the stickers, increasing viscosity and limiting its lubrication performance. This embodiment illustrates that the ibuprofen coagulated prodrug combines long-acting drug delivery and highly efficient cartilage lubrication, achieving a synergistic therapeutic effect of inhibiting inflammation and lubricating articular cartilage.

[0086] Example 11. In vivo joint retention experiment of polyethylene glycol ibuprofen aggregates

[0087] Because of the presence of enzymes in synovial fluid, such as collagenase and matrix-degrading enzymes, the components of the joint cavity are easily degraded by these enzymes. Therefore, it is crucial that suitable biological lubricants possess good resistance to enzymatic degradation. To evaluate the retention time and in vivo degradability of polyethylene glycol ibuprofen aggregates in the joint cavity of SD rats, 100 μL of Cy7 fluorescently labeled aggregates were injected into the right knee joint of SD rats (female, 10 weeks old, n=3) using a microsyringe. Successful injection into the joint cavity was confirmed by small animal in vivo imaging after injection. Fluorescence changes within the joint cavity were then monitored at corresponding time points until virtually no fluorescence signal was detected in the joint cavity.

[0088] like Figure 10 As shown, for the three aggregated prodrugs with different polyethylene glycol chain lengths, their fluorescence intensity all exhibited a similar time-dependent pattern: a rapid decrease in fluorescence intensity within one week after injection, followed by a plateau phase with a slower rate of decrease. Until day 21, a small amount of fluorescence signal could still be detected by IP400 and IP1000. This indicates that the aggregates can effectively persist within the joint cavity. Compared to easily degradable hyaluronic acid used clinically, this can reduce the frequency of injections, improve patient compliance, and demonstrate promising clinical translation potential.

[0089] Example 12. Preparation of polyethylene glycol phenylboronic acid ester aggregates

[0090] 20 ml of ultra-dry tetrahydrofuran was added to a round-bottom flask to dissolve polyethylene glycol, followed by the addition of 720 mg (30 mmol) 3 eq sodium hydride. Gas was initially generated, and an empty balloon was used to collect the gas to prevent it from being ejected from the rubber stopper. The mixture was stirred at room temperature for 24 hours. After 24 hours, the reaction system became black and viscous. 8.909 g of bromomethylphenylboronic acid ester was weighed and added to a dry beaker, followed by the addition of 20 ml of ultra-dry tetrahydrofuran. The mixture was sonicated until completely dissolved and then slowly added to the round-bottom flask using a syringe. The reaction system was observed to change from a black, viscous state to a yellow solution. The round-bottom flask was then placed at room temperature for 72 hours. After the reaction, the insoluble oils were removed by vacuum filtration. The tetrahydrofuran was then evaporated to dryness using a rotary evaporator to obtain a yellow, viscous liquid. This liquid was dissolved in methanol and then subjected to C0.05 filtration. 18 Purification was performed by column chromatography, and the solution collected from column volumes 10-13 was a polyethylene glycol phenylboronic acid (PEG) molecular solution. The PEG phenylboronic acid condensate precursor was obtained by vacuum distillation. PEG phenylboronic acid condensate monomers were added to water and vortexed thoroughly, and the phenomenon was observed. The turbidity of the solution was then characterized by UV spectrophotometry. The degradation of droplets in a 1 wt% suspension of the PEG phenylboronic acid condensate precursor was indirectly analyzed using hydrogen peroxide at concentrations of 0.1 M, 0.2 M, 0.5 M, and 1 M.

[0091] like Figure 11As shown in Figure a, polyethylene glycol phenylboronic acid ester aggregates can achieve phase separation through sufficient vortexing to form a homogeneous milky white suspension. Simultaneously, under 1M high-concentration hydrogen peroxide conditions, the suspension of 1 wt% polyethylene glycol phenylboronic acid ester aggregates changes from a turbid state to a clear and transparent solution. This indicates that under the action of ROS (hydrogen peroxide), the phenylboronic acid ester structure of the polyethylene glycol phenylboronic acid ester aggregates undergoes delamination, making it impossible for them to maintain phase separation through hydrophobic interactions. The droplets in the suspension then disappear, resulting in a clear solution, demonstrating ROS response. Figure 11 As shown in b, polyethylene glycol phenylboronic acid ester aggregates rapidly dissociate at hydrogen peroxide concentrations of 0.5M and 1M, achieving complete dissociation within 60 minutes. However, at a hydrogen peroxide concentration of 0.1M, the droplet degradation rate is significantly lower than at the 0.5M and 1M concentrations, with some droplets remaining undissociated even after 60 minutes. Phenylboronic acid esters are ROS-responsive functional groups. In the high ROS environment of tumors, they can detach from prodrugs under the influence of ROS, achieving targeted drug delivery.

[0092] The PEG modified with phenylboronic acid ester in this embodiment can also be connected to phenylboronic acid ester and its derivatives at one end and to therapeutic molecules, such as butyric acid, ibuprofen, aripiprazole, etoposide, methyltestosterone, triamcinolone, dexamethasone acetate, fluoride acetate, estradiol, cholic acid, geraniol, etc., at the other end.

[0093] Example 13. Preparation of polyethylene glycol aggregates with different end groups

[0094] 13.1 Under the action of alkaline catalysts (dicyclohexyldiimide and 4-(dimethylamino)pyridine), polyethylene glycol dicarboxylic acid (10 g) with a molecular weight of 600 and fluocinolone acetonide, triamcinolone acetonide, dexamethasone acetate, etoposide, or estradiol were added to a three-necked flask at a molar ratio of 1:3. An appropriate amount of ultra-dry dichloromethane was added, and the mixture was stirred thoroughly with a stir bar. Esterification was carried out at room temperature for 24 hours to obtain a mixture. The supernatant was filtered, recrystallized in isopropanol solution, and dried to obtain the product. The molecular structure of the product was characterized by 1H NMR spectroscopy, and the results showed that polyethylene glycol molecules with different end groups were successfully synthesized. Polyethylene glycol molecules with fluocinolone acetonide, triamcinolone acetonide, dexamethasone acetate, or etoposide end groups underwent liquid-liquid phase separation in aqueous solution under vortexing. The images observed using an optical microscope are shown below. Figure 12 As shown in the diagram, polyethylene glycol aggregates are formed; however, polyethylene glycol molecules with estradiol end groups do not undergo liquid-liquid phase separation and cannot form aggregates (as shown in the diagram). Figure 13 (as shown in b).

[0095] 1000 ml of ultra-dry tetrahydrofuran was added to a round-bottom flask, followed by 720 mg (30 mmol) of sodium hydride at 3 eq. Gas production began, and then 8.92 g of aripiprazole was added. The mixture was stirred at 60 °C for 5 h. After 5 h, the suspension was cooled to 4 °C, and 6 g of polyethylene glycol dicarboxylic acid (molecular weight 600) was added dropwise to the mixture. The reaction was allowed to proceed for 3 h to obtain the final mixture. The mixture was purified by silica gel column chromatography using a mobile phase of dichloromethane and methanol in a ratio of 98:2–80:10 (v / v). The solution collected from column volumes 3–9 was identified as a polyethylene glycol aripiprazole solution, which was then purified by vacuum distillation to obtain polyethylene glycol aripiprazole. Its molecular structure was verified by 1H NMR spectroscopy. Adding an appropriate amount of polyethylene glycol aripiprazole to water resulted in liquid-liquid phase separation, forming polyethylene glycol aripiprazole aggregates (such as...). Figure 14 (as shown in a).

[0096] 13.3 Under the action of an alkaline catalyst (dibutyltin dilaurate), polyethylene glycol (10g) with a molecular weight of 300 and butyl isocyanate or hexyl isocyanate were added to a three-necked flask at a molar ratio of 1:2. An appropriate amount of ultra-dry dichloromethane was added, and the mixture was stirred thoroughly. An amidation reaction was carried out at 60°C for 24 hours to obtain a mixture. The mixture was purified by silica gel column chromatography using a mobile phase of dichloromethane and methanol in a ratio of 98:2 to 80:10 (v / v). The solutions collected from column volumes 2 to 8 were polyethylene glycol aminobutyrate solutions or polyethylene glycol aminohexanoate solutions. These solutions were then obtained by vacuum distillation. The molecular structure was characterized by 1H NMR spectroscopy, confirming successful synthesis. When an appropriate amount of aminobutyrate or polyethylene glycol aminohexanoate was added to water, liquid-liquid phase separation occurred. Observation under an optical microscope revealed the formation of polyethylene glycol condensates (as shown in the figures below). Figure 14 (as shown in b and c).

[0097] Polydisperse polyethylene glycols with benzyl isocyanate-substituted end groups were prepared according to the above method, wherein the molecular weights of the polyethylene glycols were 600, 800, and 1000 (P600B2, P800B2, and P1000B2, respectively). The molecular weight changes of the PEGs before and after modification were analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the results are shown in Figure 15a. When the end-group-modified polyethylene glycols were added to water, the results showed that all the modified polyethylene glycols could self-assemble into aggregates (such as...). Figure 15 (as shown in b).

[0098] Monodisperse polyethylene glycols of different molecular weights were prepared according to the above method. Appropriate amounts of monodisperse polyethylene glycol molecules with different end groups and deionized water were added sequentially to a glass bottle, and the mixture was vortexed for 10 seconds to ensure homogeneity. The formation of aggregates by the monodisperse polyethylene glycol molecules with different end groups was observed. The experimental results are as follows: Figure 16 As shown.

[0099] The polyethylene glycol-based aggregates shown in the above embodiments can be prepared as prodrugs for various drugs with high safety. The preparation method of prodrugs is simple and can be industrialized. Compared with short-acting formulations, prodrugs formed by polyethylene glycol modification avoid burst release of drugs and can slowly release drugs in vivo, achieving long-acting drug delivery and improving patient compliance. In addition, compared with long-acting formulations, prodrug aggregates formed by polyethylene glycol can undergo liquid-liquid phase separation, avoiding dilution by body fluids (synovial fluid, tears, intestinal fluid, etc.), which can reduce the dosage and improve safety while maintaining drug efficacy.

[0100] Example 14 Preparation of polypropylene glycol aggregates

[0101] Using low molecular weight polypropylene glycol (PPG) as a hydrophilic polymer segment, liquid-liquid phase separation can also be driven to form PPG aggregates by introducing hydrophobic groups at both ends of the PPG molecule. In some embodiments, diamino-terminated PPG (number average molecular weight approximately 400 Da) is used, where the terminal amino groups undergo a condensation reaction with isocyanate groups, and alkyl groups are introduced at both ends of the PPG molecule as stickers via urea groups. In aqueous solution, this molecule can drive liquid-liquid phase separation through hydrogen bonding between urea groups and hydrophobic interactions between alkyl groups, thereby forming PPG aggregates.

[0102] Experimental Procedure: Synthesis of PPG-But condensate precursor: 10 mmol of diamino-terminated polypropylene glycol (PPGA, Mn400) and 20 mmol of n-butyl isocyanate were dissolved in 10 mL of N,N-dimethylformamide (DMF). Then, under magnetic stirring (300 rpm, 0℃), the n-butyl isocyanate solution was added dropwise to the PPGA solution. The reaction was continued at room temperature (25℃) with stirring for 4 hours. After the reaction was complete, the mixture was dialyzed against deionized water for 3 days using a dialysis bag (molecular weight cutoff of 500 Da) to remove residual reactants and solvents. Finally, the PPG-But condensate precursor was obtained by freeze-drying. The 1H NMR spectrum is shown below. Figure 17 As shown in a.

[0103] Synthesis of PPG-Hex condensate precursor: 10 mmol of diamino-terminated polypropylene glycol (PPGA, Mn400) and 20 mmol of n-hexyl isocyanate were dissolved in 10 mL of DMF. Under magnetic stirring (300 rpm, 0 °C), the n-hexyl isocyanate solution was added dropwise to the PPGA solution, and the reaction was continued at room temperature (25 °C) for 4 hours. After the reaction was complete, the mixture was dialyzed against deionized water for 3 days using a dialysis bag (molecular weight cutoff of 500 Da) to remove residual reactants and solvent. Finally, the PPG-Hex condensate precursor was obtained by freeze-drying. The 1H NMR spectrum is shown below. Figure 17 As shown in b.

[0104] The prepared isocyanate-modified PPG was added to water, and the self-assembled aggregates formed were as follows: Figure 18 As shown in a. The Cy3-labeled agglomerates were diluted with 1 mL of PBS to prepare agglomerate dispersion solutions. A suitable amount of the agglomerate dispersion solution was taken and a specific region of the agglomerate was fluorescently bleached using a confocal microscope. The dynamic fluorescence bleaching and recovery curves were recorded. It was observed that the fluorescence of the ROI region of the PPG agglomerates recovered in a short time after bleaching, which fully demonstrates that the agglomerates maintain the internal liquid environment (as shown in 18b).

Claims

1. A condensate formed by the self-assembly of hydrophilic polymer chains with hydrophobic end groups, characterized in that, The hydrophilic polymer chain is selected from one or more of polyethylene glycol and its block copolymers, polypropylene glycol and its block copolymers, polyacrylic acid and its block copolymers, and polyamino acids and their block copolymers. The hydrophobic group is selected from alkane groups, aromatic hydrophobic groups, or hydrophobic compounds such as aripiprazole, etoposide, methyltestosterone, triamcinolone, dexamethasone acetate, and fluocinolone acetonide.

2. The aggregate as described in claim 1, characterized in that, The degree of polymerization of the hydrophilic polymer chain is 3-20.

3. The aggregate as described in claim 1, characterized in that, The alkane group has 2-50 carbon atoms; the aromatic hydrophobic group is selected from phenylboronic acid esters and their derivatives, aromatic hydrocarbons and their derivatives, or azobenzene and its derivatives.

4. The aggregate as described in claim 1, characterized in that, The hydrophobic groups are connected to the hydrophilic polymer chain via ester bonds, amide bonds, ether bonds, or urea bonds.

5. The aggregate as described in claim 1, characterized in that, The molecular weight of the hydrophilic polymer chain is 200-1200.

6. A method for preparing an aggregate, comprising the following steps: A hydrophilic polymer chain with hydrophobic end groups is added to water, physiological solution, or other aqueous phase and self-assembles to form a condensate. The hydrophilic polymer chain is characterized by being selected from one or more of polyethylene glycol and its block copolymers, polypropylene glycol and its block copolymers, polyacrylic acid and its block copolymers, and polyamino acids and their block copolymers; the hydrophobic group is selected from alkane groups, aromatic hydrophobic groups, or hydrophobic compounds such as aripiprazole, etoposide, methyltestosterone, triamcinolone, dexamethasone acetate, and fluocinolone acetonide.

7. The preparation method according to claim 6, characterized in that, The degree of polymerization of the hydrophilic polymer chain is 3-20.

8. The preparation method according to claim 6, characterized in that, The method for preparing the hydrophilic polymer chain with the hydrophobic group-modified end group includes reacting the compound containing the hydrophobic group with the hydrophilic polymer chain and its derivatives through esterification, amidation, ureation or Williamson reaction.

9. The use of the aggregate of claim 1 in the preparation of precursor drugs and lubricants.

10. The use of the aggregate of claim 1 in drug delivery.

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