Triazole-containing polymers and methods of use thereof

Abstract

Disclosed herein are compounds of formula AL-R1(I), where these variables are defined herein, as well as medical devices comprising the compounds. The disclosure also provides pharmaceutical compositions comprising the compounds or medical devices disclosed herein. Additionally, the disclosure provides methods of treatment using the compounds, medical devices, or pharmaceutical compositions disclosed herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 210,377, filed June 14, 2021, which is incorporated by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with Government support under Grant No. R01DK120459 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] I. FIELD OF THEINVENTION The present invention relates generally to the fields of biology, chemistry and medicine, and more specifically to compounds, compositions and methods for the treatment and prevention of diseases and disorders, such as those associated with fibrosis. [Background technology]

[0004] II. Description of Related Art Diabetes, hemophilia, mucopolysaccharidoses and many other diseases can be managed or suppressed by protein drugs such as insulin or monoclonal antibodies, or the more recently emerging cell therapies. However, protein drugs cannot be administered orally and must be administered intravenously (IV). Protein drugs are rapidly degraded, limiting their therapeutic efficacy and necessitating regular IV injections to maintain therapeutic levels in the body. The burden of regular IV injections severely limits patients' quality of life and leads to high healthcare costs.

[0005] Cells transplanted into the human body could in principle act as "living factories" and constantly produce protein drugs. However, the patient's immune system destroys these foreign transplanted cells, and therefore some mechanism of encapsulating the cells in a non-immunogenic material is necessary to enable long-term cell therapy. The field of cell therapy has long been limited because biomaterials for encapsulating cells induce a foreign body reaction, i.e., fibrosis. This fibrosis envelops the graft, restricting the transfer of oxygen and nutrients to the encapsulated cells and causing cell death. There is therefore an urgent need in medicine to develop sufficiently biocompatible transplant devices or immunoprotective coatings that can prevent host immune recognition and delay fibrosis.

[0006] Previous studies have demonstrated the use of a high-throughput hydrogel library to screen a library of covalently modified triazole-containing alginate analogues to identify lead biomaterials effective in preventing fibrosis in rodent as well as non-human primate models (Vegas et al., 2016). Screening of a total of 774 combinatorially synthesized chemicals led to the identification of three lead triazole-containing anti-fibrotic small molecule compounds with similar molecular structures (Vegas et al., 2016). For in vivo evaluation of such alginate-encapsulated materials, eight different subcutaneous implantation sites were used in each mouse. However, it is important to emphasize that high-throughput screening of novel biomaterials involves the rigorous use of living subjects (mice / non-human primates: NHPs), which significantly increases the time and expense and injures a large number of rodents / non-human primates. However, the number of samples tested is very small, and a consistent high-throughput screening method has not been developed to screen a large number of immune-protective biomaterials in a single rodent / NHP, which would help reduce the number of live experimental subjects. Therefore, there is a need for additional triazole compounds as well as high-throughput screening methods to identify immune-protective biomaterials.

[0007] Development of this invention was funded in part by Cancer Prevention and Research Institute of Texas Grant No. RR160047. Summary of the Invention

[0008] The present disclosure provides triazole-containing compounds having antifibrotic properties, pharmaceutical compositions, methods for their preparation, and methods for their use.

[0009] In one aspect, the compound of the formula: AL-R1(I) or a pharma- ceutically acceptable salt thereof. During the ceremony: A is a polymer; L is of the following formula: NR a X1(CH2CH2O) o is the linker for: During the ceremony: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; o is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) Is it; or the following expression: NR b (CH2) p X2 is the linker for: During the ceremony: R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; p is 1, 2, or 3; and X2 is an arenjiil (C≦12) or substituted arenediyl (C≦12) and; R1 is cycloalkyl (C≦12) ;Haloaryl (C≦12)S-containing heteroaryl (C≦12) ;Substituted S-containing heteroaryl (C≦12) ;Alkyl (C≦6) , haloalkyl (C≦6) , alkenyl (C≦6) , or alkynyl (C≦6) Substituted Aryl (C≦12) ;Aralkyl (C≦12) ;Substituted aralkyl (C≦12) Heterocycloalkyl (C≦12) ;Substituted heterocycloalkyl (C≦12) ;2-Pyridinyl;3-Aminophenyl;4-Alkoxy (C≦6) Substituted Aryl (C≦12) ;or the following formula: X3OR2 Based on: During the ceremony: X3 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R2 is aryl (C≦12) or substituted aryl (C≦12) That is, A compound, or a pharma- ceutically acceptable salt thereof, is provided.

[0010] In some embodiments, the compound is further defined as follows, or a pharma- ceutically acceptable salt thereof: AL-R1(I) During the ceremony: A is a polymer; L is of the following formula: NR a X1(CH2CH2O) m is the linker for: During the ceremony: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; m is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R1 is cycloalkyl (C≦12) ;Haloaryl(C≦12) S-containing heteroaryl (C≦12) ;Substituted S-containing heteroaryl (C≦12) ;Alkyl (C≦6) , haloalkyl (C≦6) , alkenyl (C≦6) , or Alkinie (C≦6) Substituted Aryl (C≦12) ;3-Aminophenyl;4-Alkoxy (C≦6) Substituted Aryl (C≦12) ;or the following formula: X3OR2 Based on: During the ceremony: X3 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R2 is aryl (C≦12) or substituted aryl (C≦12) It is.

[0011] In some embodiments, the compound is further defined as follows, or a pharma- ceutically acceptable salt thereof: AL-R1(I) During the ceremony: A is a polymer; L is of the following formula: NR a X1(CH2CH2O) m is the linker for: During the ceremony: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; m is 2, 3, 4, or 5; and X1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) Is it; or the following expression: NR b (CH2) n X2 is the linker for: During the ceremony: R b is hydrogen, alkyl (C≦6) , or substituted alkyl(C≦6) and; n is 1, 2, or 3; and X2 is an arenjiil (C≦12) or substituted arenediyl (C≦12) and; R1 is haloaryl (C≦12) ;Aralkyl (C≦12) ;Substituted aralkyl (C≦12) Heterocycloalkyl (C≦12) ;Substituted heterocycloalkyl (C≦12) ;2-pyridinyl;3-aminophenyl.

[0012] In some embodiments, the polymer comprises one or more saccharide repeat units, e.g., the repeat units have the following formula: has TIFF2024522379000001.tif26128, During the ceremony: R3 or R4 are each independently hydrogen or hydroxy; R5 is hydroxy, alkoxy (C≦8) , substituted alkoxy (C≦8) or a covalent bond to a linker; and m is the number of repeating units having a molecular weight of about 50,000 daltons to about 500,000 daltons.

[0013] In some embodiments, the polymer has the following formula: TIFF2024522379000002.tif contains 33128 repeat units, During the ceremony: R3, R3', R4, or R4' are each independently hydrogen or hydroxy; R5 is hydroxy, alkoxy (C≦8) , substituted alkoxy (C≦8) or a covalent bond to said linker; R5' is a covalent bond to the linker; and m and n will be the number of repeating units having a molecular weight of about 50,000 daltons to about 500,000 daltons.

[0014] In some embodiments, the polymer is an acrylate polymer, such as a methacrylate polymer. In some embodiments, o is 2 or 3. In some embodiments, o is 2. In other embodiments, o is 3. In some embodiments, R a is hydrogen. In some embodiments, X1 is an alkanediyl, such as -CH2CH2-. (C≦6) In some embodiments, R b is hydrogen. In some embodiments, p is 1 or 2. In some embodiments, p is 1. In some embodiments, X2 is an arenediyl, such as benzenediyl. (C≦12) It is.

[0015] In some embodiments, R1 is a haloaryl, such as chlorophenyl, bromophenyl, or fluorophenyl. (C≦12) In some embodiments, R1 is 2-bromophenyl, 4-chlorophenyl, 2-fluorophenyl, or 4-fluorophenyl. In other embodiments, R1 is 3-aminophenyl. In other embodiments, R1 is an S-containing heteroaryl. (C≦12) or substituted S-containing heteroaryl (C≦12) In some embodiments, R1 is an S-containing heteroaryl, such as 2-thiophenyl or 3-thiophenyl. (C≦12) In other embodiments, R1 is cycloalkyl, such as cyclopropyl. (C≦12) In another embodiment, R1 is alkyl. (C≦6) , haloalkyl (C≦6) , alkenyl (C≦6) , or Alkinie (C≦6) Substituted Aryl (C≦12) In some embodiments, R1 is an alkyl group, such as 3-methylphenyl or 4-methylphenyl. (C≦6) Substituted Aryl (C≦12) In other embodiments, R1 is haloalkyl, such as 4-trifluoromethylphenyl. (C≦6) Substituted Aryl (C≦12) In other embodiments, R1 is an alkyne group such as 3-ethyne-phenyl. (C≦6) Substituted Aryl(C≦12) In some embodiments, R1 has the following formula: X3OR2 Based on During the ceremony: X3 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) and; R2 is aryl (C≦12) or substituted aryl (C≦12) It is.

[0016] In some embodiments, X3 is an alkanediyl, such as -CH2-. (C≦8) In some embodiments, R2 is a substituted aryl, such as 4-aminophenyl. (C≦12) In other embodiments, R1 is 4-alkoxy, such as 4-ethoxyphenyl. (C≦6) Substituted Aryl (C≦12) In another embodiment, R1 is heterocycloalkyl. (C≦12) or substituted heterocycloalkyl (C≦12) In some embodiments, R1 is a heterocycloalkyl, such as thiomorpholine-dioxide. (C≦12) In another embodiment, R is aralkyl. (C≦12) or substituted aralkyl (C≦12) In some embodiments, R1 is an aralkyl group, such as 2-phenylethyl. (C≦12) In some embodiments, the compound is: TIFF2024522379000003.tif208105TIFF2024522379000004.tif231114TIFF2024522379000005.tif234125, During the ceremony m and n will be the number of repeating units having a molecular weight of about 50,000 daltons to about 500,000 daltons.

[0017] In yet another aspect, the present disclosure provides a method of detecting fibrosis in a sample, the method comprising exposing the sample to one or more polymers described herein and measuring reactivity.

[0018] In another aspect, the present disclosure provides a medical device that is coated with the compound described herein.In some embodiments, the medical device is implantable device, cardiac pacemaker, catheter, needle injection catheter, thrombus filter, vascular graft, balloon, stent graft, biliary stent, intestinal stent, bronchial stent, esophageal stent, ureteral stent, aneurysm filling coil or other coil device, surgical repair mesh, breast implant, silicone implant, PDMS, transmyocardial revascularization device, percutaneous myocardial revascularization device, prosthesis, organ, blood vessel, aorta, heart valve, tube, organ replacement part, implant, fiber, hollow fiber, membrane, fabric, blood deposit, blood container, titer plate, adsorption medium, dialyzer, connection part, sensor, valve, endoscope, filter, pump chamber, or other medical device that is intended to have blood compatibility, or that is used in cancer, diabetes, ischemia, antibacterial, hemophilia, stroke, blood disorder, or cytokine therapy that includes human modified cells.

[0019] In some embodiments, the medical device is a capsule, an implantable polymer block, a 3D printed block, a 3D printed gel, or a polymer encapsulation device. In some embodiments, the polymer encapsulation device further comprises a shape selected from a sphere, a square, a noodle, a needle, a rectangle, and a cylinder. In some embodiments, the implantable capsule is a microcapsule. In some embodiments, the medical device is a catheter. In some embodiments, the medical device is less fibrotic than an uncoated medical device. In some embodiments, the medical device is immunoprotective compared to an uncoated medical device. In some embodiments, the immunoprotective results in a lower foreign body reaction.

[0020] In yet another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (A) a compound or medical device described herein; and (B) Excipients The present invention provides a pharmaceutical composition comprising:

[0021] In some embodiments, the pharmaceutical composition further comprises a biological material. In some embodiments, the biological material is encapsulated in the compound or medical device. In some embodiments, the biological material is a cell. In some embodiments, the cell is a cell from a heterologous tissue, a cell from a cadaver, a stem cell, a cell derived from a stem cell, a cell from a cell line, a primary cell, a reprogrammed cell, a reprogrammed stem cell, a cell derived from a reprogrammed stem cell, a genetically engineered cell, or a combination thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is an insulin-producing cell. In some embodiments, the cell is a pancreatic islet cell. In some embodiments, the compound is crosslinked. In some embodiments, the crosslinked compound is covalently crosslinked.

[0022] In yet another aspect, the present disclosure provides a method for treating or preventing disease or disorder, comprising administering to a patient in need thereof a compound, medical device, or pharmaceutical composition as described herein.In some embodiments, the method results in lower foreign body reaction.In some embodiments, the method results in less fibrosis.

[0023] Other objects, features and advantages of the present disclosure will become clear from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description, it should be understood that this detailed description and specific examples, while showing specific embodiments of the present invention, are presented for illustrative purposes only. It should be noted that simply because a specific compound is assigned to one specific general formula, it does not mean that it cannot also be assigned to another general formula. [Brief description of the drawings]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figure 1 shows a general schematic outlining the synthesis of new alginate analogs. The specific linkers and alkynes used are listed in Table 2. On the left side of the scheme are two of the previous triazole-containing lead alginates (B1-A21 and Z1-A34) that prevent fibrosis in mouse and NHP models. A total of 211 new alginate analogs were synthesized by varying the lead hydrophilic linker (azido-PEG-amine) and hydrophobic linker (iodobenzylamine) in combination with a series of alkyne classes shown in the center. [Diagram 2]Figures 2A-2D show the cell barcoding strategy for material screening. (Figure 2A) Overall schematic: 20 different HUVEC donors were encapsulated with the corresponding materials and implanted in mice for 4 weeks to evaluate the antifibrotic properties and biocompatibility. (Figure 2B) 20 unique HUVECs were sequenced by NGS to identify their specific SNPs, which can be used as barcodes to tag and identify different encapsulation materials in vivo. (20) Unique HUVECs were sequenced by next generation sequencing (NGS) to identify their individual single nucleotide polymorphisms (SNPs), which can be used as barcodes to tag and identify different encapsulation materials in vivo. (Figure 2C, 2D) To prove that all donors (H1-H20) could be deconvoluted by NGS, a mixture of 20 different capsules encapsulating 20 different HUVEC donors was implanted in NSG mice for 4 weeks. (Figure 2C) Bright-field and dark-field images before and after implantation: Capsules were retrieved with minimal cell deposition, demonstrating the absence of fibrosis, similar to capsules before implantation. Encapsulated cells remain viable 4 weeks after implantation (live cells: green, dead cells: red). Scale bar, 2 mm. (Figure 2D) Capsules from one mouse were analyzed by NGS, successfully identifying 195 capsules out of 200. Across donors, the percentage of identified capsules is evenly distributed from donor 1 to donor 20. [Diagram 3] Figures 3A-3E show the gelation assay results and material characterization. (Figure 3A) Gelation assay of alginate analogues using entrapment of Rhodamine B. (Figure 3B) Representative images of capsule formation using alginate analogues. Scale bar, 2 mm. (Figure 3C) After initial characterization tests including purity, solubility, and gel-forming ability, 149 alginate polymers were used in screening tests. (Figure 3D) Table of elemental analysis of alginate linkers confirming attachment of linkers to the alginate backbone. (Figure 3E) Representative NMR images showing triazole peaks of alginate analogues confirming modification. [Figure 4]Figures 4A-4C show the optimization of DNA extraction method by pre-implantation capsules. The optimized method was used for further analysis such as qPCR and NGS identification by post-implantation capsules. (Figure 4A) Comparison of DNA content by capsule dissolution conditions; with or without EDTA dissolution step and in fresh vs. frozen state. (Figure 4B) Comparison of DNA content by DNA elution conditions; elution volume and elution temperature. (Figure 4C) Comparison of DNA content / yield by cell number per capsule. [Diagram 5] Figures 5A and 5B show the optimization of the NGS library preparation workflow. Library preparation includes a multiplex PCR step to amplify SNP loci, a barcode PCR step to add position barcodes to each sample, and a ligation-based sequence adapter correction step. (Figure 5A) Before optimization, the on-target rate of the libraries was <10%, with primer dimers and nonspecific PCR products dominating the read data. (Figure 5B) After optimization, the on-target rate increased to >80%, despite the low DNA input (<1 ng) in the starting material. [Figure 6]Figure 6 shows the bioinformatics pipeline for determining material identity / composition from NGS sequencing data. Fastq NGS data were demultiplexed by row and column barcodes to realign sequences amplified from the same DNA input. For each amplicon sequence, the grep function was then applied to search for dominant and mutant alleles, and the mutant allele frequency (VAF) of each SNP locus was calculated. If the encapsulated cells contained only one donor, the VAF profile was compared to the profiles of 20 pre-screened HUVEC donors. The donor that showed the highest concordance rate was identified as the encapsulated donor cells. If one or two donors were used as encapsulated cells, the log-likelihood of all possible donor compositions was calculated. The composition with the highest overall log likelihood was determined as the cell composition (quality controls for log likelihood analysis: 1) at least 25 / 30 SNP loci had sequence coverage of >50; and 2) overall log likelihood >-200, and 3) goodness measure >10, where goodness is defined as the difference in log likelihood between the most likely and second most likely donor pair). The material corresponding to the identified donor cells or cell composition is the material that encapsulates the cells. [Figure 7]Figures 7A-7F show that high-throughput screening of combinatorially synthesized chemically modified alginates with unique cell barcoding facilitates the identification of novel hydrogels with reduced fibrosis in immune-competent mice. (Figure 7A) A library of immune-modulating biomaterials; a total of 211 novel alginate analogs were synthesized. (Figure 7B) To increase the throughput of screening, a mixture of different materials was implanted into the same implantation site. Material identity was determined by demultiplexing the SNP genotypes of encapsulated HUVECs using an NGS assay. A typical implantation of 200 alginate capsules (approximately 1.5 mm in diameter, 10 capsules / material) allows for the simultaneous evaluation of 20 different implantation materials, with enough independent capsules per material to allow statistical analysis. (Figure 7C) Representative results from one of the rounds. Four weeks after implantation, the capsules were excised. Transparent capsules with reduced fibrosis (bottom row), similar to pre-implantation capsules, were isolated for further analysis. Scale bar, 10 mm. (Figure 7D) Heat map summarizing the material screening of all alginate analogues. (Figure 7E) 149 new materials were screened and the corresponding lead materials were identified by NGS assay. Error bars represent 95% confidence intervals from a binomial distribution. The mean level of Z1-A34 (previous positive materials, shown as bars) is shown as a dotted line. The top materials ranking above Z1-A34 are shown as dark bars. (Figure 7F) Representative structures of the top three lead alginate analogues (orange bars in Figure 7E). [Figure 8]Figures 8A-8H show the scale-up of material screening in the NHP model using dual donor barcoding. (Figure 8A) Two HUVEC donors were mixed at a 1:2 ratio and encapsulated in various materials. (Figure 8B) With 20 HUVEC donors mixed at a 1:2 ratio, 20 x 20 = 400 different SNP profiles were generated. (Figure 8C) 100 donor pairs were encapsulated with the corresponding materials and implanted into the IP cavity of NHPs for 4 weeks. 30 capsules per material were used, for a total of 3000 capsules were implanted. (Figure 8D) Representative image of capsules before implantation. (Figure 8E) After 4 weeks, all floating capsules in the IP cavity were collected and used for material identification (light arrows: capsules with fibrotic tissue aggregates, grey arrows: floating capsules). (Figure 8F) Summary of donor pair identification. Of the total 503 selected capsules, 466 (92.6%) were identified with high confidence, 32 (6.36%) were identified with low confidence, and 5 (0.99%) capsules failed to be identified. (Figure 8G) Distribution of confidence levels of analyzed capsules. Goodness is the difference in log-likelihood between the most likely and the second most likely donor pair, therefore, a high goodness indicates a low probability of misidentification. Capsules in the upper right corner are more confident, while those with a log-likelihood below -200 or a goodness below 10 are considered "low confidence". (Figure 8H) Chemical structures of the top 4 identified leads. [Figure 9] Figures 9A-9D show optimization of barcoding of two HUVEC donors to expand barcoding capacity for larger library screening. (Figure 9A) Three different mixtures of materials containing corresponding donor pairs were tested in vitro. The goodness is the difference in log likelihood between the most likely pair chosen and the second most likely pair, which is a measure of how good the chosen combination is. (Figures 9B-9D) Representative heatmaps plot the log likelihood of each of 20 x 20 donor combinations with different mixing ratios (b, 1:2; c, 1:3; d, 1:4). The density of each small box represents the likelihood. [Figure 10]Figures 10A-10F show dual donor barcoding identification in C57BL / 6J mice. (Figure 10A) Three different materials were tested; UP-VLVG (control), B1-A51 (one of the negative materials), and Z1-A34 (one of the positive materials). (Figure 10B) Schematic workflow of three materials screening including mixed dual donors. (Figures 10C and 10D) Two weeks after transplantation, capsules were collected from each mouse (M1-M3) and divided into three groups according to fibrosis levels. (Figure 10E) Representative heatmap results of identified donor pairs. (Figure 10F) Thirty-nine samples were mapped to Z1-A34 (positive control material) encoded by H16:H14 in a 1:2 ratio; four were mapped to UP-VLVG encoded by H6:H8 in a 1:2 ratio; 0 samples were mapped to B1-A51. Overall, 43 / 45 samples were mapped from 400 donor SNP profiles. The proportions of each sample corresponding to the donor pair were plotted, with Z1-A34 showing the highest value and demonstrating the best immune protection. [Figure 11] Figures 11A-11C show that lead hydrogels exhibit low fibrosis in the peritoneal cavity of C57BL / 6J mice. (Figure 11A) Representative darkfield images of excised microcapsules (300-400 μm in size) retrieved from the IP cavity before and 2 weeks after implantation. Scale bar, 2 mm. (Figure 11B) Representative confocal images of excised microcapsules; capsules were stained with CD68 (light), DAPI (gray), and α-SMA (dark) markers. (Figure 11C) RT-qPCR analysis to compare RNA expression in different materials. Expression of fibrosis markers (α-SMA and Col1a1) was normalized to SLG20 (control). Statistical analysis was performed using two-way ANOVA with Bonferroni correction (****P<0.0001, SLG20 control vs. others). [Figure 12]Figure 12 shows the results of a diabetes reversal study with the lead material (Z4-A10). Capsules containing human islets were made with final cell densities of 4,000, 8,000, and 16,000 IEQ / mL of alginate. The final IEQ values ​​for each capsule were 10, 20, and 40 IEQ / capsule, respectively. In each group, 500 μL, 250 μL, and 125 μL capsules containing a total of 2,000 IEQ per mouse were implanted into the IP cavity. [Figure 13] Figures 13A-13H show that lead hydrogels encapsulating xenogeneic human islets exhibit diabetes reversal in immunocompetent C57BL / 6J mice. (Figure 13A) Representative images of capsules before implantation. Z4-A10 capsules containing human islets at densities of 10 IEQ / capsule, 20 IEQ / capsule, and 40 IEQ / capsule, respectively. SLG20 capsules were used as control material. Dithizone staining shows viable islets within the capsule matrix. After encapsulation, the islets showed good viability (live: light, dead: dark). (Figure 13B) Blood glucose levels in Z4-A10 and SLG20 groups (4,000 IEQ / mL density) were monitored until the mice were euthanized (****P<0.0001 (SLG20 vs. Z4-A10)). (Figure 13C) IVGTT study of Z4-A10 capsule (4,000 IEQ / mL) implanted group in diabetic mice and non-implanted group in diabetic and non-diabetic mice (ns; not significant, ****P<0.0001 (all comparisons)). (Figure 13D, Figure 13E) Representative dark field (Figure 13D) and dithizone stained (red, Figure 13E) images of excised Z4-A10 and SLG20 capsules (4,000 IEQ / mL). (Figure 13F) Human c-peptide measurements (SLG20 vs. Z4-A10) 80 days after implantation. (Figure 13G, Figure 13H) Blood glucose monitoring in high islet density groups: Z4-A10 capsule (Figure 13G) and SLG20 capsule (Figure 13H). Error bars indicate mean ± sem; two-way ANOVA with Bonferroni multiple comparison correction. [Figure 14]14A-14E show that lead immunoprotective small molecules were used to coat catheter tubing to provide immune protection in the subcutaneous space of C57BL / 6 mice. (FIG. 14A) Chemical structure of unmodified or Met-Z1-A3 or Met-B2-A17 coated groups. (FIG. 14B) XPS data of unmodified, Met-Z1A3, and Met-B2-A17 modified catheters showing the weight percent of small molecule specific atoms, indicating successful coating. (FIG. 14C) ToF-SIMS data of unmodified, Met-Z1A3, and Met-B2-A17 modified catheters showing the area of ​​normalized intensity (au) by total ion intensity of major peaks (CN-, Br-) indicating successful coating. (FIG. 14D) Representative histology of fibrotic capsule measured for unmodified and coated catheters. Thin, low density purple band of cells at the tissue-catheter interface of coated catheters indicates milder immune response. (FIG. 14E) Quantification of fibrotic capsule thickness in unmodified and coated catheters. Statistical analysis was by one-way ANOVA with Bonferroni correction (****P<0.0001, ***P<0.002). [Figure 15] Figures 15A-15E show material characterization and evaluation of catheters coated with lead molecules. (Figure 15A, Figure 15B) The summed intensity of the two main peaks analyzed by Tof-SIMS (Figure 15A, CN- and Figure 15B, Br-) was plotted and compared to unmodified catheters. (Figure 15C) Representative SEM images of unmodified and coated catheters. (Figure 15D) Examples of deposited fibrotic capsule tissue measured. Using ImageJ, tissue deposition was measured by purple bands of tissue adjacent to the catheter. (Figure 15E) Representative H&E stained sections of excised catheters from each group. Scale bar, 2 mm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Description of exemplary embodiments Disclosed herein are new compounds and compositions with antifibrotic properties, their preparation methods, and their use, including for the treatment and / or prevention of disease.These compounds are alginate derivatives (referred to as modified alginate compounds) that contain one or more triazole groups that link the compounds to the alginate backbone.These compounds have one or more improved properties, such as improved compatibility or activity in in vivo or in vitro assays, compared to other alginates known in the art.

[0026] The present invention describes small molecules and small molecule-polymer conjugates that (1) have antifibrotic properties and (2) maintain the viability of encapsulated cells. The chemical structures of these materials are based on antifibrotic small molecules identified through an initial screening of approximately 700 materials (Vegas et al., 2016). Previous studies suggest that the identified triazole compounds with immunomodulatory properties likely occupy a special structural space where the immunomodulatory performance could not be predicted without performing the screening described herein. The triazole-containing modifications associated with improved in vivo performance collectively suggest that structural analogs around these triazole modifications may be a versatile chemical space for designing biomaterials that can mitigate foreign body reactions and modulate immune responses.

[0027] I. Compounds of the Invention The compounds of the present invention (also referred to as "modified alginate compounds", "compounds of the present disclosure" or "compounds disclosed herein") are shown, for example, in the Summary of the Invention section above and in the claims below. They can be made using the synthetic methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry applied by those of skill in the art. Such principles and techniques are taught, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated herein by reference. In addition, the synthetic methods can be further modified and optimized for preparative, pilot or large-scale production, either batch or continuous, using the principles and techniques of process chemistry applied by those of skill in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development - A Guide for Organic Chemists (2012), which is incorporated herein by reference.

[0028] Table 1. Examples of modified alginate compounds described herein TIFF2024522379000006.tif235152TIFF2024522379000007.tif222152TIFF2024522379000008.tif224152TIFF2024522379000009.tif50152

[0029] All of the compounds of the present invention may, in some embodiments, be used in the prevention and treatment of one or more diseases or disorders discussed herein or elsewhere. In some embodiments, one or more of the compounds characterized or exemplified herein as intermediates, metabolites, and / or prodrugs may nevertheless also be useful in the prevention and treatment of one or more diseases or disorders. Thus, unless expressly stated to the contrary, all of the compounds of the present invention are considered to be "active compounds" and "therapeutic compounds" intended for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting public health by ensuring the safety, efficacy, quality, and security of drugs, vaccines and other biological products, and medical devices for human and veterinary use.

[0030] In some embodiments, the compounds of the present invention have the advantage that they may be more effective, less toxic, longer acting, more potent, have fewer side effects, be more readily absorbed, be metabolically stable, be lipophilic, be hydrophilic, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art, whether for use in the indications described herein or otherwise.

[0031] The compounds of the present invention may contain one or more asymmetrically substituted carbon, sulfur, or phosphorus atoms and may be isolated in optically active or racemic forms. Therefore, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of the chemical formula are intended, unless a specific stereochemistry or isomeric form is specifically indicated. The compounds may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention may have an S or R configuration. In some embodiments, the compounds of the present invention may contain multiple atoms with a defined stereochemical orientation.

[0032] The chemical formulas used to represent the compounds of the present invention typically only show one of several different possible tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which tautomer is most prevalent, all tautomers of a given chemical formula are intended.

[0033] In addition, the atoms constituting the compounds of the present invention are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 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, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 140, 141, 152, 16 13 C and 14 Contains C.

[0034] In some embodiments, the compounds of the present invention function as prodrugs or can be derivatized to function as prodrugs. Prodrugs are known to enhance many desirable properties of drugs (e.g., solubility, bioavailability, manufacture, etc.), so the compounds used in some methods of the present invention may be delivered in prodrug form if desired. Thus, the present invention contemplates prodrugs of the compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds used in the present invention may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved to the parent compounds, either by routine manipulation or in vivo. Thus, prodrugs include compounds described herein, for example, where a hydroxy group, an amino group, or a carboxy group is bonded to any group that is cleaved to form a hydroxy, amino, or carboxylic acid, respectively, when the prodrug is administered to a patient.

[0035] In some embodiments, the compounds of the present invention can be in salt or non-salt form.With respect to salt form, in some embodiments, the specific anion or cation that forms part of any salt form of the compounds provided herein is not important, as long as the salt as a whole is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and their preparation and use are shown in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0036] It will be understood that many organic compounds can form complexes with the solvents in which they react or from which they precipitate or crystallize. These complexes are known as "solvates". When the solvent is water, the complexes are known as "hydrates". It will also be understood that many organic compounds can exist in multiple solid forms, including crystalline and amorphous forms. All solid forms of the compounds provided herein, including any solvates thereof, are within the scope of the present invention.

[0037] II. Use of the Compound The modified alginates described herein may be used in a variety of applications in the food, pharmaceutical, cosmetic, agricultural, printing, and textile industries. Alginates are widely used in the food industry as thickening, gelling, stabilizing, thickening, suspending, and emulsifying agents. Alternatively, these modified alginates may be used as matrices to control the delivery of therapeutic, preventive, and / or diagnostic agents. In addition, these modified alginates may be incorporated into pharmaceutical compositions as excipients, where they may act as thickening, suspending, emulsifying, binding, and disintegrating agents. Modified alginates may also be used in other applications, such as dental impression materials, components of wound dressings, and printing agents. Those skilled in the art will recognize that the modified alginates disclosed herein may be used in any application in which any currently used alginate or modified alginate may currently be used. It is specifically contemplated that the modified alginates described herein may be used in applications where improved biocompatibility and physical properties (e.g., anti-fibrotic properties) as compared to commercially available alginates are preferred.

[0038] i. Cell encapsulation Historically, alginates, and thus the modified alginates described herein, can be ionically crosslinked with divalent cations in water at room temperature to form a hydrogel matrix. See, for example, U.S. Patent No. 4,352,883. In this method, an aqueous solution containing the biological material to be encapsulated is suspended in a solution of a water-soluble polymer, the suspension is formed into droplets, which are contacted with polyvalent cations to form discrete capsules, and the capsule surface is then crosslinked with polyamino acids to form a semipermeable membrane around the encapsulated material.

[0039] Water-soluble polymers having charged side groups are crosslinked by reacting them with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups, or multivalent anions if the polymer has basic side groups. Cations for crosslinking polymers having acidic side groups to form hydrogels are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, but also alkyl ammonium salts, e.g., Di-, tri-, or tetrafunctional organic cations such as TIFF2024522379000010.tif4128 can also be used. Aqueous solutions of salts of these cations are added to the polymer to form soft, highly swollen hydrogels and membranes. The higher the concentration of the cation or the higher the valency, the higher the degree of crosslinking of the polymer. It has been shown to crosslink polymers from concentrations as low as 0.005M. Higher concentrations are limited by the solubility of the salt.

[0040] Anions for crosslinking polymers containing basic side chains to form hydrogels are divalent and trivalent anions such as low molecular weight dicarboxylic acids, e.g., terephthalic acid, sulfate, and carbonate. Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels and membranes as described for the cations.

[0041] A variety of polycations can be used to complex and thereby stabilize the polymer hydrogel into a semipermeable surface membrane. Examples of materials that can be used include polymers with basic reactive groups, such as amine or imine groups, with molecular weights between 3,000 and 100,000, such as polyethyleneimine and polylysine. These are commercially available. One polycation is poly(L-lysine); examples of synthetic polyamines are: polyethyleneimine, poly(vinylamine), and poly(allylamine). There are also natural polycations, such as the polysaccharide chitosan.

[0042] Polyanions that can be used to form semipermeable membranes by reaction with basic surface groups on polymer hydrogels include polymers and copolymers of acrylic acid, methacrylic acid, and other acrylic acid derivatives, polymers with pendant SO3H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.

[0043] In one embodiment, the cells are encapsulated in a modified alginate polymer. In one embodiment, modified alginate capsules are made from a solution of modified alginate containing suspended cells using an encapsulating agent (e.g., Inotech® encapsulating agent). In some embodiments, the modified alginate is encapsulated using Ca 2+ , B.A. 2+ or Sr 2+ In some embodiments, the modified alginate is cross-linked with a multivalent cation such as BaCl2. In some embodiments, the capsule is further purified after formation. In some embodiments, the capsule is washed with, for example, HEPES solution, Krebs solution, and / or RPMI-1640 medium.

[0044] The cells can be obtained directly from a donor, from cell culture of cells from a donor, or from an established cell culture line. In some embodiments, the cells are obtained directly from a donor, washed, combined with a polymeric material, and directly implanted. The cells are cultured using techniques known to those skilled in the art of tissue culture. In some embodiments, the cells are autologous, i.e., derived from the individual into whom the cells are implanted, but may also be allogeneic or xenogeneic.

[0045] Cell adhesion and viability can be assessed using scanning electron microscopy, histology, and quantitative radioisotope assessment. The function of transplanted cells can be determined using a combination of the techniques and functional assays mentioned above. For example, in the case of hepatocytes, in vivo liver function tests can be performed by cannulating the recipient's common bile duct. A small amount of bile can then be collected. Bile pigments can be analyzed by high pressure liquid chromatography for underivatized tetrapyrroles, or by reaction with diazotized azodipyrrole ethyl anthranilate, with or without P-glucuronidase treatment, to convert them to azodipyrroles, followed by thin layer chromatography. Diconjugated and monoconjugated bilirubin can also be measured by thin layer chromatography after alkaline methanolysis of conjugated bile pigments. Generally, as the number of functioning transplanted hepatocytes increases, so do the levels of conjugated bilirubin. Simple liver function tests, such as albumin production, can also be performed on blood samples. Similar organ function tests can be performed using techniques known to those skilled in the art, as needed to determine the degree of cell function after transplantation. For example, pancreatic islet cells can be delivered in a manner similar to that used specifically to transplant hepatic cells to achieve glucose regulation by proper secretion of insulin to cure diabetes. Other endocrine tissues can also be transplanted. Tests using labeled glucose as well as tests using protein assays can be performed to quantify the cell mass on the polymer scaffold. These cell mass tests can then be linked to cell function tests to determine the appropriate cell mass. In the case of chondrocytes, function is defined as providing the appropriate structural support to the surrounding adherent tissue.

[0046] This technology can be used to provide multiple cell types, including genetically modified cells, within a three-dimensional scaffold to efficiently engraft large numbers of cells and promote graft survival for the purpose of generating new tissue or tissue equivalents, and can also be used to immune protect the cell graft while the new tissue or tissue equivalent is growing by excluding the host immune system.

[0047] Examples of cells that can be transplanted as described herein include chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells.As used herein, "organ cells" include liver cells, pancreatic islet cells, intestinal cells, kidney cells, and other cells that act primarily to synthesize and secrete materials or metabolize.A particular cell type is pancreatic islet cells.

[0048] The polymeric matrix can be combined with humoral factors to promote cell implantation and engraftment, for example, the polymeric matrix can be combined with angiogenic factors, antibiotics, anti-inflammatory agents, growth factors, differentiation-inducing compounds, and other factors known to those skilled in the art of cell culture.

[0049] For example, humoral factors can be mixed in a sustained release form with the cell-alginate suspension prior to forming an implant for implantation, or the hydrogel can be modified to bind humoral factors or signal recognition sequences prior to combination with the isolated cell suspension.

[0050] The techniques described herein can be used to deliver many different cell types to achieve different tissue structures. In one embodiment, the cells are mixed with a hydrogel solution and injected directly into the site where it is desired to implant the cells before the hydrogel hardens. However, the matrix can be molded and implanted into one or more different sites of the body to suit a specific application. This application is particularly suitable when a specific structural design is desired or when the area where the cells are to be implanted does not have a specific structure or support to promote cell growth and proliferation.

[0051] The site or sites for implanting the cells are determined based on individual needs, as well as the number of cells required. For cells with organ functions, such as hepatocytes or pancreatic islet cells, the mixture can be injected into the mesentery, subcutaneous tissue, retroperitoneum, preperitoneal space, and intramuscular space. For cartilage formation, the cells are injected into the site where cartilage formation is desired. A mold can also be applied externally to mold the injected solution. In addition, by controlling the polymerization rate, it is possible to mold the cell-hydrogel injected implant like molding clay. Alternatively, the mixture can be injected into a mold, the hydrogel can be hardened, and then the material can be implanted.

[0052] ii. Coating Products and Surfaces Medical products can be coated with the disclosed modified alginate polymers using a variety of techniques, examples of which include spraying, dipping, and brushing. Polymer coatings are typically applied to the surface to be coated by dissolving the polymer in a suitable organic solvent and applying it by spraying, brushing, dipping, painting, or other similar techniques. The coating agent is deposited on the surface and bonds to the surface via non-covalent interactions. The resulting coated products and surfaces are specifically contemplated and disclosed.

[0053] In some embodiments, the surface may be pretreated with an appropriate solution or suspension to modify the properties of the surface, thereby enhancing non-covalent interactions between the modified surface and the coating.

[0054] The polymer solution is applied to a surface at an appropriate temperature for a sufficient time to form a coating on the surface, where the coating is effective to form an antifibrotic surface. Typical temperatures include room temperature, but higher temperatures may be used. Typical times include 5 minutes or less, 30 minutes or less, 60 minutes or less, and 120 minutes or less. In some embodiments, the solution may be applied for 120 minutes or more to form a coating with desired antifibrotic activity. However, shorter times may be used. Antifibrotic activity can be measured by any of the methods disclosed herein or known in the art. Antifibrotic activity can be a foreign body reaction determined as described herein.

[0055] The modified alginate compounds described herein can be covalently or non-covalently attached to products, devices, and surfaces. In those embodiments in which the modified alginate compounds described herein are covalently attached to products, devices, or surfaces, the polymer can be linked to the product, device, or surface by, for example, functionalizing the product, device, or surface with a reactive functional group, such as a nucleophilic group, and reacting the nucleophilic group with a reactive functional group on the polymer, such as an electrophilic group. Alternatively, the polymer can be functionalized with a nucleophilic group, and reacting with an electrophilic group on the product, device, or surface.

[0056] In certain embodiments, the modified alginate compounds described herein are non-covalently bound to the product, device, or surface. The polymer can be applied to the product, device, or surface by spraying, wetting, dipping, soaking, painting, gluing or attaching, or by otherwise providing the modified alginate compounds described herein to the product, device, or surface. In one embodiment, the polymer is applied by spraying, painting, or dipping or soaking. For example, a polymer coating can be prepared by dissolving the modified alginate compounds described herein in a suitable solvent (typically aqueous) and optionally sonicating the solution so that the polymer is completely dissolved. The product, device, or surface to be coated can be immersed in the polymer solution for a suitable time, for example, 5 seconds, followed by drying, such as air drying. This procedure can be repeated as many times as necessary to achieve sufficient coverage. The thickness of the coating is generally about 1 nm to about 1 cm, preferably about 10 nm to 1 mm, more preferably about 100 nm to about 100 microns.

[0057] The coating can be applied at the time the product, device, or surface is manufactured, or after the product, device, or surface is manufactured. In some embodiments, the coating is applied to the product, device, or surface immediately prior to use of the product, device, or surface.

[0058] This is called intraoperative coating.As used herein, "immediately before" means within 1, 2, 5, 10, 15, 20, 30, 45, 60, 75, 90, 120, 150, 180 minutes or more before implantation or use.In some embodiments, the product, device or surface is coated in hospital, for example, in operating room, within 20, 15, 10 or 5 minutes before implantation or use.Coating immediately before use can overcome the limitations of products, devices and surfaces coated at the time of manufacture, such as damage to the coating during storage and / or transportation of the product, device or surface, and / or the effectiveness of the coating decreases over time when the coating is exposed to harsh environmental conditions (e.g., exposure to high temperature, humidity, ultraviolet light, etc.).

[0059] The coated medical products can be used for the known applications and purposes of uncoated medical products or medical products with different coatings.

[0060] A. Medical Products Medical products useful for coating include any type of medical device that is used, at least in part, for implantation into a patient's body. Examples include implants, implantable medical products, implantable devices, catheters and other tubes (including urinary and biliary tubes, endotracheal tubes, wound drainage tubes, needle injection catheters, peripherally inserted central catheters, dialysis catheters, long term tunneled central catheters peripheral venous catheters, short term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urethral catheters, peritoneal catheters), vascular catheter ports, thrombus filters, urinary tract devices (including long term urinary tract devices, tissue bound urinary tract devices, artificial urinary sphincters, urinary dilators), shunts (including ventricular or arteriovenous shunts, stent grafts, biliary stents, intestinal stents, bronchial stents, esophageal stents, urethral stents, and hydrocephalus shunts), balloons, pacemakers, implantable cardioverter defibrillators, orthopedic devices, and the like. These include, but are not limited to, medical products (including pins, plates, screws, and implants), grafts (including organs, vascular grafts, blood vessels, aortas, heart valves, and organ replacement parts), prostheses (including breast implants, penile prostheses, vascular graft prostheses, heart valves, artificial joints, artificial larynxes, otic implants, artificial hearts, artificial blood vessels, and artificial kidneys), aneurysm filling coils and other coil devices, transmyocardial revascularization devices, percutaneous myocardial revascularization devices, tubes, fibers, hollow fibers, membranes, blood containers, titer plates, adsorption media, dialyzers, connections, sensors, valves, endoscopes, filters, pump chambers, scalpels, needles, scissors (and other devices used in invasive surgical, therapeutic, or diagnostic procedures), and other medical products and devices intended to have antifibrotic properties. The term "medical product" is broad and specifically refers to products that come into contact with blood either briefly (e.g., endoscopes) or permanently (e.g., stents).

[0061] Useful medical products are balloon catheters and endovascular prostheses, especially stents. A stent of normal design has a filigree support structure made up of metallic struts. This support structure is initially provided in an unexpanded state for insertion into the body and then expanded to an expanded state at the application site. The stent can be coated before or after being crimped onto the balloon. A wide variety of medical endoprostheses or medical products or implants are known for very different applications. They are used, for example, to support blood vessels, hollow organs, and ductal systems (endovascular implants), to mount and temporarily attach tissue implants and tissue grafts, and for orthopedic purposes such as pins, plates, or screws.

[0062] The modified alginate compounds described herein can be applied, absorbed, or attached to a variety of different substrates and surfaces. Examples of suitable materials include metals, metallic materials, ceramics, polymers, fibers, inert materials such as silicon, and combinations thereof.

[0063] Suitable polymeric materials include, but are not limited to, styrene and substituted styrenes, ethylene, propylene, poly(urethanes), acrylates and methacrylates, acrylamides and methacrylamides, polyesters, polysiloxanes, polyethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), copolymers thereof, and combinations thereof.

[0064] The substrate may be in the form of, or form part of, a film, particle (nanoparticle, microparticle, or millimeter sized beads), fiber (wound dressings, bandages, gauze, tape, pads, sponges, such as woven and nonwoven sponges, and those specifically designed for dental or ophthalmic surgery), sensor, pacemaker lead, catheter, stent, contact lens, bone implant (artificial hip joints, pins, rivets, plates, bone cement, etc.), or tissue regeneration or cell culture device, or other medical device for use within or in contact with the body.

[0065] Described herein are implants coated with modified alginate compound coatings. An "implant" is any object that is not living tissue and is intended to be placed inside a mammalian body, such as a human. An implant is a form of medical product. Implants include naturally occurring objects that have been processed to kill their living tissue. As an example, a bone graft can be processed to remove its living cells but maintain its shape to serve as a template for ingrowth of bone from a host. As another example, natural coral can be processed to obtain hydroxyapatite preparations that can be applied to the body for certain orthopedic and dental treatments. An implant is also an article that includes an artificial component. The term "implant" can apply to the full range of medical devices intended for placement inside a human or mammalian body, including orthopedic, dental, otorhinolaryngological ("ENT"), and cardiovascular applications.

[0066] In some embodiments, "implant" as used herein refers to a macroscopic composition that includes an implantation device or the surface of an implantation device and a modified alginate compound coating.In these embodiments, the term "implant" does not include nanoparticles and / or microparticles.As used herein, "macroscopic" generally refers to a device, implant, or composition that can be seen by the naked eye.

[0067] Examples of implantable medical devices and medical devices and mechanical structures in which biocompatible coatings may be used include, but are not limited to, stents, conduits, scaffolding, heart valve rings, cardiovascular valves, pacemakers, artificial hip joint devices, implantable sensor devices, esophageal stents, cardiac implants, biocompatible linings for heart valves, oxygen delivery tubing for dialysis machines and cardiopulmonary bypass systems.

[0068] In general, a stent is a device, typically tubular in shape, that is inserted into a body lumen, such as a blood vessel or duct, to prevent or offset a localized flow restriction. The purpose of a stent is to mechanically support and open a body fluid duct in some cases. Stents are often used to mitigate reduced blood flow to organs and limbs to maintain adequate delivery of oxygenated blood. The most common use of stents is in the coronary arteries, but they are also widely used in other body ducts, such as central and peripheral arteries and veins, bile ducts, esophagus, colon, trachea, large bronchi, ureters, and urethra. Often, stents inserted into a lumen are expandable or self-expanding after insertion. For example, metal stents are placed into a blocked artery using a balloon catheter and expanded to restore blood flow. For example, stainless steel wire mesh stents are commercially available from Boston Scientific, Natick, Mass.

[0069] In some embodiments, the implant is an orthopedic implant. An "orthopedic implant" is defined as an implant that replaces or provides fixation to bone, replaces a joint surface, provides an abutment for a prosthesis, or a combination thereof, or assists in replacing or providing fixation to bone, replacing a joint surface, providing an abutment for a prosthesis, and combinations thereof.

[0070] Orthopedic implants can be used to replace or provide fixation to bone, replace articulating surfaces of joints, provide abutments for prostheses, or combinations thereof, or to assist in replacing or providing fixation to bone, replacing articulating surfaces of joints, providing abutments for prostheses, including dental applications, and combinations thereof.

[0071] Suitable orthopedic implants include, but are not limited to, wires, Kirschner wires, bone plates, screws, pins, tacks, rods, nails, nuts, bolts, washers, spikes, buttons, wires, fracture plates, reconstruction and stabilizer devices, intracorporeal and extracorporeal prostheses (articulating and non-articulating), intraosseous percutaneous prostheses, spacers, meshes, implant abutments, anchors, barbs, clamps, sutures, interbody fusion devices, tubes of any shape, scaffoldings, and combinations thereof.

[0072] In other embodiments, the implant is an ear, nose and throat ("ENT") implant. Exemplary ENT implants include, but are not limited to, Eustachian tubes, endotracheal tubes, ventilation tubes, cochlear implants, and bone-anchored hearing devices.

[0073] In other embodiments, the implant is a cardiovascular implant. Exemplary cardiovascular implants include heart valves or alloplastic vessel wall supports, total artificial heart implants, ventricular assist devices, vascular grafts, stents, electrical signal transmission devices such as pacemakers and neural leads, defibrillator leads, and the like.

[0074] Implants can be prepared from various materials. In some embodiments, the material is biocompatible. In some embodiments, the material is biocompatible and non-biodegradable. Exemplary materials include metal materials, metal oxides, polymeric materials, including degradable and non-degradable polymeric materials, ceramics, porcelain, glass, allogeneic, xenogeneic bone or bone matrix; genetically engineered bone; and combinations thereof.

[0075] Suitable metallic materials include, but are not limited to, titanium-based metals and alloys (e.g., nitinol, nickel-titanium alloys, thermal memory alloy materials), stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chromium, or cobalt-chromium and cobalt-chromium-nickel alloys, such as certain cobalt alloys, including ELGILOY® and PHYNOX®.

[0076] Useful examples include stainless steel grade 316 (SS 316 L) (consisting of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2% Mn, <1% Si, <0.45% P, and <0.03% S), tantalum, chromium-molybdenum alloys, nickel-titanium alloys (e.g., Nitinol), and cobalt-chromium alloys (e.g., MP35N, ASTM material designation: 35Co-35Ni-20Cr-10Mo). Typical metals currently used in stents include SS 316 L steel and MP35N. See also, ''Comparing and Optimizing Co-Cr Tubing for Stent Applications,'' Poncin, P, Millet, C., Chevy, J, and Profit, JL, Materials & Processes for Medical Devices Conference, August 2004, ASM International.

[0077] Suitable ceramic materials include, but are not limited to, oxides, carbides, or nitrides of transition elements such as titanium oxide, hafnium oxide, iridium oxide, chromium oxide, aluminum oxide, and zirconium oxide. Silicon-based materials such as silica may also be used.

[0078] Suitable polymeric materials include polystyrene and substituted polystyrenes, polyethylene, polypropylene, polyacetylene, polystyrene, TEFLON®, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethanes), polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, polyesters, polysiloxanes, polyethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), silicones, epoxy resins, Kevlar®, Dacron®, and the like. Polyolefins (condensation polymers derived from ethylene glycol and terephthalic acid), nylons, polyalkenes, phenolics, natural and synthetic elastomers, adhesives and sealants, biopolymers such as polyolefins, polysulfones, polyacrylonitriles, polysaccharides and natural latex, collagen, cellulose polymers (e.g., alkylcelluloses, etc.), polysaccharides, poly(glycolic acid), poly(L-lactic acid) (PLLA), polydioxanone (PDA), or racemic poly(lactic acid), polycarbonates, (e.g., polyamides (nylons); fluoroplastics, carbon fibers, and blends or copolymers thereof.

[0079] The polymer may be covalently or non-covalently bound to the surface, although in certain embodiments, the polymer is non-covalently bound to the surface. The polymer may be applied by a variety of techniques in the art, including, but not limited to, spraying, wetting, immersion, immersion such as dip coating (e.g., intraoperative dip coating), painting, or otherwise applying a hydrophobic, polycationic polymer to the surface of the implant.

[0080] Surfaces of products adapted for use in a healthcare environment are capable of being sterilized using gas treatment techniques such as autoclaving, biocide exposure, irradiation, or ethylene oxide exposure. Surfaces found in healthcare environments include the interior and exterior aspects of various instruments and devices, whether for single use or repeated use.

[0081] b. Hydrogel Medical products can be made from or use hydrogels. The modified alginate compounds described herein can form hydrogels for this and other purposes. Products made with other hydrogels can also be coated with the modified alginate polymers disclosed. Thus, the modified alginate compounds described herein can be used as a coating on a product or surface, or as a product itself. Hydrogels are three-dimensional hydrophilic polymer networks that can absorb large amounts of water or biological fluids (Peppas et al., Eur. J. Pharm. Biopharm. 2000, 50, 27-46). These networks consist of homopolymers or copolymers and are insoluble due to chemical or physical crosslinks, e.g., entanglements or the presence of crystallites. Hydrogels can be classified as neutral or ionic based on the nature of the side groups. In addition, they may be amorphous, semi-crystalline, hydrogen-bonded structures, supramolecular structures and hydrocolloid aggregates (Peppas, NA Hydrogels. In: Biomaterials science: an introduction to materials in medicine; Ratner, BD, Hoffman, AS, Schoen, FJ, Lemons, JE, Eds; Academic Press, 1996, pp. 60-64; Peppas et al., Eur. J. Pharm. Biopharm. 2000, 50, 27-46). Hydrogels may be prepared from synthetic or natural monomers or polymers. Hydrogels may include modified alginate compounds as described herein.

[0082] Hydrogels can be prepared from synthetic polymers such as poly(acrylic acid) and its derivatives [e.g., poly(hydroxyethyl methacrylate) (pHEMA)], poly(N-isopropylacrylamide), poly(ethylene glycol) (PEG) and its copolymers, and poly(vinyl alcohol) (PVA), among others (Bell and Peppas, Adv. Polym. Sci. 122:125-175 (1995); Peppas et al., Eur. J. Pharm. Biopharm. 50:27-46 (200); Lee and Mooney, Chem. Rev. 101:1869-1879 (2001)). Hydrogels prepared from synthetic polymers are generally non-degradable under physiological conditions. Hydrogels can also be prepared from natural polymers, including, but not limited to, polysaccharides, proteins, and peptides. The modified alginate compounds described herein are an example. These networks are generally degraded by chemical or enzymatic means under physiological conditions.

[0083] In some embodiments, the hydrogel is non-degradable under relevant in vitro and in vivo conditions. Stable hydrogel coatings are necessary for certain applications, including central venous catheter coatings, heart valves, pacemakers and stent coatings. In other cases, hydrogel degradation may be a preferential approach, for example, in tissue engineering constructs.

[0084] In some embodiments, hydrogels can be formed by dextran. Dextran is a bacterial polysaccharide that consists essentially of α-1,6 linked D-glucopyranose residues with a few percent of α-1,2, α-1,3, or α-1,4 linked side chains. Dextran is widely used for biomedical applications due to its biocompatibility, low toxicity, relatively low cost, and simple modification. This polysaccharide has been used clinically for over 50 years as a plasma expander, peripheral blood flow enhancer, and antithrombotic agent (Mehvar, RJ Control. Release 2000, 69, 1-25). In addition, dextran has been used as a polymeric carrier for the delivery of drugs and proteins, primarily to extend the life of therapeutic agents in the circulation. Dextran can be modified with vinyl groups by either chemical or enzymatic means to prepare gels (Ferreira et al. Biomaterials 2002, 23, 3957-3967).

[0085] Dextran-based hydrogels prevent adhesion of endothelial cells, smooth muscle cells, and fibroblasts (Massia, SP; Stark, JJ Biomed. Mater. Res. 2001, 56, 390-399. Ferreira et al. 2004, J. Biomed. Mater. Res. 68A, 584-596), and dextran surfaces prevent protein adsorption (Osterberg et al., J. Biomed. Mat. Res. 1995, 29, 741-747).

[0086] As described herein, the modified alginate compounds described herein can be used to encapsulate cells. In some embodiments, the encapsulated cells can be fabricated into a macrodevice. For example, in some embodiments, the cells encapsulated in the modified alginate hydrogel can be coated onto a surface, such as a flat surface. In some embodiments, the capsules containing the cells can be attached to the tissue of a subject using a biocompatible adhesive. In other embodiments, the capsules containing the cells can be coated onto a medical device suitable for implantation.

[0087] iii. Treatment of a disease or disorder Alternatively, the encapsulated cells can be implanted into a patient in need thereof to treat a disease or disorder. In some embodiments, the encapsulated cells are obtained from a non-genetically identical member of the same species. In alternative embodiments, the encapsulated cells are obtained from a different species than the patient. In some embodiments, hormone or protein secreting cells are encapsulated and implanted into a patient to treat a disease or disorder.

[0088] In some embodiments, the disease or disorder is caused by or involves dysfunctional hormone or protein secreting cells in the patient.In some embodiments, the disease or disorder is diabetes.The medical products, devices, and surfaces coated with the modified alginate compounds described herein can be implanted or embedded in patients in need of the same to treat the disease or disorder.The capsules, products, devices, and surfaces disclosed can continue to show substantially no fibrotic effects or low foreign body reaction for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or more after administration or implantation.

[0089] The disclosed capsules, products, devices, and surfaces can be administered or implanted alone or in combination with any suitable drug or other therapy. Such drugs and therapies can also be administered separately (i.e., used in parallel) while the capsules, products, devices, and surfaces are present in the patient. The disclosed capsules, products, devices, and surfaces reduce fibrosis and immune reactions to the capsules, products, devices, and surfaces, but do not preclude the use of anti-inflammatory drugs and immune system suppressants together with or in parallel with the capsules, products, devices, and surfaces. However, in one embodiment, the disclosed capsules, products, devices, and surfaces are used without the use of anti-inflammatory drugs and immune system suppressants. In some embodiments, fibrosis remains reduced even after the use, concentration, effect, or combination of any anti-inflammatory or immune system suppressant used falls below effective levels. For example, fibrosis may remain reduced for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, or more after the use, concentration, effect, or combination thereof of any anti-inflammatory or immune system suppressant used has fallen below effective levels.

[0090] III. Combination Therapy In addition to being used as a monotherapy, the compound of the present invention can also be used in combination with one or more other therapies.Effective combination therapy can be achieved with a single composition or pharmacological formulation that contains both drugs, or with two different compositions or formulations that are administered simultaneously, one composition containing the compound of the present invention and the other containing the second drug.Alternatively, the therapy can be administered before or after the other drug treatment, with intervals ranging from minutes to months.

[0091] Non-limiting examples of such combination therapies include the combination of one or more compounds of the present invention with another anti-inflammatory agent, a chemotherapeutic agent, radiation therapy, an antidepressant, an antipsychotic agent, an anticonvulsant, a mood stabilizer, an anti-infective agent, an antihypertensive agent, a cholesterol-lowering agent or other blood lipid regulating agent, an agent that promotes weight loss, an antithrombotic agent, an agent for treating or preventing a cardiovascular event such as myocardial infarction or stroke, an antidiabetic agent, an agent that reduces transplant rejection or graft-versus-host disease, an anti-arthritic agent, an analgesic agent, an anti-asthmatic agent or other treatment for a respiratory disease, or an agent for treating or preventing a skin disorder.

[0092] IV. Definition The following definitions supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be taken to indicate that any term not defined is indefinite. Rather, all terms used are believed to describe the invention in such terms that one skilled in the art can understand the scope and practice the invention.

[0093] When used in the context of chemical groups, "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written -COOH or -CO2H); "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means =NH; "cyano" means -CN; "Isocyanyl" means -N=C=O; "azido" means -N3; ​​in the monovalent context "phosphate" means -OP(O)(OH)2 or its deprotonated form; in the divalent context "phosphate" means -OP(O)(OH)O- or its deprotonated form; "mercapto" means -SH; and "thio" means =S; "thiocarbonyl" means -C(=S)-; "sulfonyl" means -S(O)-; and "sulfinyl" means -S(O)-.

[0094] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. The symbol "----" represents any bond, if present, which may be either single or double. The symbol TIFF2024522379000011.tif4128 represents a single or double bond. Therefore, the formula For example, TIFF2024522379000012.tif11128 is TIFF2024522379000013.tif11128. It is understood that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereoatoms. Instead, it encompasses all stereoisomers as well as mixtures thereof. The symbol TIFF2024522379000014.tif4128 shows the bond (e.g., methyl When drawn vertically across a 3D image (TIFF2024522379000015.tif7128), it indicates the point of attachment of the group. Note that attachment points are typically only identified in this fashion for larger groups to help the reader clearly identify the point of attachment. TIFF2024522379000016.tif4128 denotes a single bond in which the group attached to the thick end of the wedge is "off the page". TIFF2024522379000017.tif4128 represents a single bond in which the group attached to the thick end of the wedge is "into the page". TIFF2024522379000018.tif4128 refers to a single bond where the geometry around the double bond (e.g., either E or Z) is undefined. Thus, both options as well as combinations thereof are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is pointing out of the plane of the paper.

[0095] The variable may be a "floating group" on the ring system, such as in the formula: When depicted as a group "R" in TIFF2024522379000019.tif15128, that variable may replace any hydrogen atom attached to any ring atom, including drawn, implied, or explicitly defined hydrogens, so long as a stable structure is formed. When depicted as a group "R" in TIFF2024522379000020.tif18128, that variable may replace any hydrogen attached to any ring atom of any of the fused rings, unless otherwise noted. Substitutable hydrogens include drawn hydrogens (e.g., hydrogens attached to the nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens attached to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present in either the 5-membered or 6-membered ring of the fused ring system. The subscript "y" immediately following the parenthesized R in the formula above represents a numerical variable. Unless otherwise noted, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substitutable hydrogen atoms in the ring or ring system.

[0096] For chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" or "C=n" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that may be present in the group / class, with the minimum number being as small as possible for that group / class. For example, the group "alkyl (C≦8) ", "Alkandiyl (C≦8) ", "Heteroaryl (C≦8) " and "Acyl (C≦8) The minimum number of carbon atoms in the group "alkenyl" is 1. (C≦8) ", "alkynyl (C≦8) " and "heterocycloalkyl (C≦8) The minimum number of carbon atoms in the group "cycloalkyl" is two.(C≦8) The minimum number of carbon atoms in the group "aryl" is 3. (C≦8) " and "Arengeiil (C≦8) The minimum number of carbon atoms in the group is 6. "Cn-n'" defines both the minimum (n) and the maximum (n') number of carbon atoms in the group. Thus, "alkyl (C2-10) " denotes an alkyl group having from 2 to 10 carbon atoms. These carbon number designators may precede or follow the chemical group or class that they modify, and may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C 1-4 -alkyl", "C1-4-alkyl", "alkyl (C1-4) " and "Alkyl (C≦4) " are all synonymous. Except as noted below, all carbon atoms are counted to determine whether a group or compound falls within a specified number of carbon atoms. For example, a dihexylamino group is a dialkylamino group. (C12) Examples of groups include dialkylamino. (C6) Similarly, phenylethyl is not an example of an aralkyl group. (C=8) is an example of a substituted alkyl group. When any of the chemical groups or compound classes defined herein are modified with the term "substituted," any carbon atoms in the moiety that replace a hydrogen atom are not counted. Thus, methoxyhexyl, which has a total of 7 carbon atoms, is a substituted alkyl group. (C1-6) Unless otherwise stated, any chemical group or compound class recited in a claim without a carbon atom limit has a carbon atom limit of 12 or less.

[0097] The term "saturated", when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. When such bonds are present, they do not exclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism. When the term "saturated" is used to modify a solution of a substance, it means that no more of the substance can be dissolved in that solution.

[0098] The term "aliphatic" means that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, compound or group. In an aliphatic compound / group, the carbon atoms can be linked in straight chains, branched chains, or non-aromatic rings (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) linked by single carbon-carbon bonds, or unsaturated with one or more carbon-carbon double bonds (alkene / alkenyl) or with one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0099] The term "aromatic" means that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n+2 electrons in a completely conjugated cyclic π system. An aromatic compound or chemical group may be depicted as a single resonance structure; however, the depiction of one resonance structure is considered to refer to any other resonance structures. For example: TIFF2024522379000021.tif16128 TIFF2024522379000022.tif16128. Aromatic compounds may be drawn using circles to represent the delocalization of electrons in fully conjugated cyclic π-systems, two non-limiting examples of which are shown below: TIFF2024522379000023.tif12128

[0100] The term "alkyl" refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment and a straight or branched acyclic structure and no atoms other than carbon and hydrogen. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or t Bu), and -CH2C(CH3)3 (neopentyl) are non-limiting examples of alkyl groups. The term "alkanediyl" refers to a divalent saturated aliphatic group with one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. The term "alkylidene" refers to the divalent group =CRR', where R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl as defined above.

[0101] The term "cycloalkyl" refers to a monovalent saturated aliphatic group having a carbon atom as a point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to a carbon atom of a non-aromatic ring structure. The term "cycloalkanediyl" refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Group TIFF2024522379000024.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula HR, where R is cycloalkyl as defined above.

[0102] The term "alkenyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It should be noted that although the alkenediyl group is aliphatic, it is not excluded that when attached at both ends, the group forms part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene with only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule.

[0103] The term "alkynyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched chain acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, where R is alkynyl.

[0104] The term "aryl" refers to an unsaturated aromatic group having an aromatic carbon atom as an attachment point, the carbon atom forming part of one or more aromatic ring structures each having six ring atoms, all of which are carbon, and the group is composed of no atoms other than carbon and hydrogen. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. As used herein, the term aryl does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and biphenyl (e.g., 4-phenylphenyl). The term "arenediyl" refers to a divalent aromatic group having two aromatic carbon atoms as attachment points, the carbon atoms forming part of one or more six-membered aromatic ring structures each having six ring atoms, all of which are carbon, and the divalent group is composed of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. When multiple rings are present, the rings may be fused or non-fused. Non-fused rings are linked by covalent bonds. Non-limiting examples of arenediyl groups include: Contains TIFF2024522379000025.tif34141.

[0105] "Arene" refers to the class of compounds having the formula H-R, where R is aryl as defined above. Benzene and toluene are non-limiting examples of arenes.

[0106] The term "aralkyl" refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the above definitions. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0107] The term "heteroaryl" refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, each of which forms part of one or more aromatic ring structures having from 3 to 8 ring atoms, at least one of the ring atoms of the aromatic ring structure being nitrogen, oxygen, or sulfur, and the heteroaryl group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When multiple rings are present, the rings are fused rings, but the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (where the carbon number limit permits) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the attachment point. "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. The term "heteroarenediyl" refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, each forming part of one or more aromatic ring structures having 3 to 8 ring atoms, at least one of the ring atoms of the aromatic ring structure being nitrogen, oxygen, or sulfur, and the divalent group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When multiple rings are present, the rings are fused rings, but the term heteroarenediyl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitations permitting) attached to one or more ring atoms. Non-limiting examples of heteroarenediyl groups include: Contains TIFF2024522379000026.tif16128.

[0108] The term "heteroaralkyl" refers to the monovalent group -alkanediyl-heteroaryl, where the terms alkanediyl and heteroaryl are each used in a manner consistent with the above definitions. Non-limiting examples are: pyridylmethyl and 2-quinolinyl-ethyl.

[0109] The term "heterocycloalkyl" refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom each forming part of one or more non-aromatic ring structures having 3 to 8 ring atoms, at least one of the ring atoms of the non-aromatic ring structure being nitrogen, oxygen or sulfur, and the heterocycloalkyl group being composed of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. When multiple rings are present, the rings are fused rings. As used herein, the term does not preclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to one or more ring atoms. The term also does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the point of attachment. Non-limiting examples of N-heterocycloalkyl groups include N-pyrrolidinyl and Contains TIFF2024522379000027.tif7128.

[0110] The term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, or aryl as defined above. The groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH(CH3), -C(O)CH(CH2), -C(O)CH5, and -C(O)CH4CH3 are non-limiting examples of acyl groups. "Thioacyl" is defined in an analogous manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkyl group, as defined above, attached to a -CHO group.

[0111] The term "alkoxy" refers to the group -OR, where R is alkyl as defined above. Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), or -OC(CH3)3 (tert-butoxy). The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The terms "alkylthio" and "acylthio" refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms is replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group.

[0112] The term "alkylamino" refers to the group -NHR, where R is alkyl as defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3. The term "dialkylamino" refers to the group -NRR', where R and R' can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: -N(CH3)2 and -N(CH3)(CH2CH3). The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl as defined above. A non-limiting example of an amido group is -NHC(O)CH3.

[0113] When a chemical group is used with the modifier "substituted," one or more hydrogen atoms of the group are, independently in each occurrence, replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CO2CH2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: -CHOH, -CHCl, -CF, -CHCN, -CHC(O)OH, -CHC(O)OCH, -CHC(O)NH, -CHC(O)CH, -CHOCH, -CHOC(O)CH, -CHNH, -CHN(CH), and -CHCHCl. ​​The term "hydroxyalkyl" is a subset of substituted alkyl in which one or more hydrogen atoms are replaced with a hydroxy (i.e., -OH) group, such that no other atoms are present other than carbon, hydrogen, and oxygen. The groups -CHOH, -CHCHOH, -CH(OH)CHOH, -CHCH(OH)CH, and -CH(OH)CHOH are non-limiting examples of hydroxyalkyl groups. The term "monohydroxyalkyl" is a subset of substituted alkyl in which one hydrogen atom is replaced with a hydroxy (i.e., -OH) group, such that there are no other atoms other than carbon, hydrogen, and one oxygen. The groups -CHOH, -CHCHOH, and -CHCH(OH)CH are non-limiting examples of monohydroxyalkyl groups. The term "fluoroalkyl" is a subset of substituted alkyl in which one or more hydrogen atoms are replaced with fluoro, such that there are no other atoms other than carbon, hydrogen, and fluorine. The groups -CHF, -CHF, and -CF are non-limiting examples of fluoroalkyl groups. The term "monofluoroalkyl" is a subset of substituted alkyl in which one hydrogen atom is replaced with fluoro, such that there are no other atoms other than carbon, hydrogen, and one fluorine. The groups -CHF, -CHCHF, and -CHCH(F)CH are non-limiting examples of monofluoroalkyl groups.The term "aminoalkyl" is a subset of substituted alkyl where one or more hydrogen atoms are replaced with an amino (i.e., -NH2) group, such that there are no other atoms other than carbon, hydrogen, and nitrogen. The groups -CH2NH2, -CH(NH2)CH3, -CH2CH2NH2, -CH2CH(NH2)CH3, and -CH(NH2)CH2NH2 are non-limiting examples of aminoalkyl groups. The term "monoaminoalkyl" is a subset of substituted alkyl where one hydrogen atom is replaced with an amino (i.e., -NH2) group, such that there are no other atoms other than carbon, hydrogen, and one nitrogen. The groups -CH2NH2, -CH2CH2NH2, and -CH2CH(NH2)CH3 are non-limiting examples of monoaminoalkyl groups. Non-limiting examples of substituted aralkyl are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl. The groups, -C(O)CHCF, -COH (carboxyl), -COCH (methylcarboxyl), -COCHCH, -C(O)NH (carbamoyl), and -CON(CH) are non-limiting examples of substituted acyl groups. The groups -NHC(O)OCH and -NHC(O)NHCH are non-limiting examples of substituted amide groups.

[0114] In the claims and / or specification, the use of the words "a" or "an" in conjunction with the term "comprising" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0115] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the device or the method being used to determine the value, or the variation that exists among test subjects or patients.

[0116] An "active ingredient" (AI) or pharma- ceutical active ingredient (API) (also called an active compound, active substance, activator, drug, agent, biologically active molecule, or therapeutic compound) is a component of a pharmaceutical agent that is biologically active.

[0117] The terms "comprise," "having," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," "including," etc., are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also includes other steps not listed.

[0118] The term "effective" as used herein and / or in the claims means sufficient to achieve a desired, expected, or intended result. An "effective amount", "therapeutically effective amount" or "pharmaceutical effective amount", when used in the context of treating a patient or subject with a compound, means an amount of the compound sufficient to treat or prevent a disease, as defined below, when administered to the patient or subject.

[0119] An "excipient" is a pharma- ceutically acceptable substance that is formulated with the active ingredient of a drug, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk the composition (and thus are often called "bulking agents," "fillers," or "diluents" when used for this purpose), or to impart therapeutic enhancements to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include anti-adherents, binders, coating agents, colorants, disintegrants, flavorings, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles of pharma- ceutically acceptable types. The primary excipient that serves as a vehicle to carry the active ingredient is usually referred to as the vehicle. Excipients may also be used in the manufacturing process to aid in the handling of the active ingredient, such as by promoting powder flowability or non-adhesiveness, in addition to aiding in vitro stability, such as preventing denaturation or aggregation during the expected storage period. The suitability of an excipient typically varies depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0120] The term "hydrate," when used as a modifier to a compound, means that in a solid form of the compound, etc., each compound molecule has less than one (e.g., a hemihydrate), one (e.g., a monohydrate), or more than one (e.g., a dihydrate) water molecule associated with it.

[0121] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit half of a given biological, biochemical or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor or microorganism).

[0122] An "isomer" of a first compound is another compound whose molecules contain the same constituent atoms as the first compound, but which differ in the arrangement of those atoms in three dimensions.

[0123] The term "patient" or "subject" as used herein refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0124] "Pharmaceutically acceptable," as generally used herein, refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / loss ratio.

[0125] "Pharmaceutically acceptable salt" refers to a salt of a compound disclosed herein that is pharma- ceutically acceptable and has the desired pharmacological activity, as defined above. Such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethane ... Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as sulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butyl acetic acid, trimethyl acetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present can react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be understood that the particular anion or cation forming a part of any salt of the present invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharma-ceutically acceptable salts and their methods of preparation and use are described in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0126] A "pharmaceutically acceptable carrier", "drug carrier", or simply "carrier" is a pharma- ceutically acceptable substance formulated with an active ingredient drug that is involved in the carrying, delivery, and / or transport of a chemical agent. Drug carriers may be used to improve drug delivery and efficacy, including, for example, controlled release technologies to modulate drug bioavailability, reduce drug metabolism, and / or reduce drug toxicity. Some drug carriers may enhance the efficacy of drug delivery to a particular target site. Examples of carriers include: liposomes, microspheres (e.g., made of polylactic-co-glycolic acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes, and dendrimers.

[0127] A "pharmaceutical drug" (also called a drug, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, agent, medication, drug therapy, medicine, or simply drug, agent, or preparation) is a composition used in the diagnosis, cure, treatment, or prevention of disease and contains a pharmacologic active ingredient (API) (defined above) and optionally one or more inactive ingredients, also called excipients (defined above).

[0128] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the pathology or symptoms of the disease, and / or (2) delaying the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the pathology or symptoms of the disease.

[0129] "Prodrug" refers to a compound that can be converted metabolically in vivo to a pharmacologic active ingredient of the present invention. The prodrug itself may or may not have activity in its prodrug form. For example, a compound containing a hydroxy group may be administered as an ester that is converted to the hydroxy compound by hydrolysis in vivo. Non-limiting examples of suitable esters that can be converted to the hydroxy compound in vivo include acetate, citrate, lactate, phosphate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-β-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinoate, and esters of amino acids. Similarly, compounds containing an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

[0130] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms but differ in the arrangement of those atoms in three dimensions. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also called a stereogenic center or stereoisomeric center, which is any point in a molecule that has groups such that any two groups, if swapped, result in a stereoisomer, not necessarily an atom. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, although other atoms can be stereogenic centers in organic and inorganic compounds. Molecules can have multiple stereocenters, giving many stereoisomers. In compounds where stereoisomerism is due to tetrahedral stereocenters (e.g., tetrahedral carbons), the total number of hypothetical possible stereoisomers is 2. nn is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum number of possible stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched, such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or axis of chirality where the stereochemistry is not defined, it is contemplated that the stereocenter or axis of chirality can exist as its R-form, S-form, or a mixture of R- and S-forms, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer.

[0131] "Treatment" or "treating" includes (1) inhibiting a disease (e.g., arresting further progression of the pathology and / or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, (2) ameliorating a disease (e.g., reversing the pathology and / or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, and / or (3) causing any measurable reduction in the disease or its symptoms in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease.

[0132] The term "unit dose" refers to a formulation of a compound or composition such that the formulation is prepared in a manner sufficient to provide a patient with a therapeutically effective dose of the active ingredient in a single administration. Such unit dose formulations that can be used include, but are not limited to, a single tablet, capsule, or other oral formulation, or a single vial containing an injectable liquid or other injectable formulation.

[0133] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that certain terms are defined should not be taken to indicate that any term not defined is indefinite. Rather, all terms used are considered to describe the invention in terms that allow one skilled in the art to understand the scope and practice the invention. EXAMPLES

[0134] V. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the invention and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0135] Example 1: Experimental Procedures and Characterization Data A. Novel biomaterials library based on alginate derivatives A combinatorial library of hydrogels was developed using genetic barcoding techniques to identify materials that reduce cognition in a preclinical fibrosis model using C57BL / 6 mice. Although the physicochemical parameters governing antifibrotic properties are not fully understood at present, a combinatorial biomaterial screening approach was developed to generate a library of alginate-based hydrogels by utilizing several reverse chemical reactions to covalently modify the functional groups and properties latent in the polymeric alginate backbone. This synthetic chemistry scheme allows for the rapid production of a stereospecific set of diverse chemical structures, and the reactions are compatible with the modification of alginate compounds. Using low molecular weight, ultra-pure VLVG alginate with high guluronic acid content (Nova Matrix inc.) as a starting material, the synthesis of a 6872-member alginate analogue library with various amines, alcohols, azides, and alkynes is proposed. In the initial phase, a total of 211 alginate analogues were synthesized by maintaining the triazole ring throughout. Of the 211 unique alginate analog polymers, approximately 150 analogs were generated from combinatorial synthesis of three hydrophilic PEG-based linkers with 51 new alkynes similar to reported hydrophilic leads (Z1-A34), and 61 triazole-containing analogs were generated from two hydrophobic linkers similar to hydrophobic leads (B1-A21) (Figure 1, Table 2). The covalently bound triazole-containing alginate analogs were characterized using elemental analysis and nuclear magnetic resonance spectroscopy (NMR) (Figure 3D, 3E). After confirmation of synthesis, purity, solubility, and gel-forming ability (gelation assay), 149 alginate analogs were selected for in vivo screening purposes (Figure 3). Elemental analysis indicated that with optimized reaction conditions, up to 20-30% of the alginate could be modified to allow significant tuning of material properties.

[0136] Novel high-throughput in vivo screening methods were developed in both rodent models that utilize xenotransplantation of human cells into C57BL / 6 model rodents undergoing fibrosis to test multiple biomaterials at a single implantation site using high-throughput biomaterial barcoding and analysis. These methods involve labeling each biomaterial with barcoded cells. A barcoding technique was developed using 20 different unique HUVECs, which was used to in vivo screen a large library of hydrogel biomaterials containing immunoprotective chemically modified triazoles in mouse and NHP models using next-generation sequencing (NGS). See Figure 2A.

[0137] Table 2: A table of the linkers and alkynes used and their designations. TIFF2024522379000028.tif205140

[0138] Table 3. Information on 20 different HUVEC donors TIFF2024522379000029.tif122166

[0139] Table 4. Synthetic alginate analogs and in vivo (mouse and NHP) screening. TIFF2024522379000030.tif102128TIFF2024522379000031.tif172128

[0140] B. Materials and Methods Reagents. All chemicals were obtained from Sigma Aldrich, USA (unless otherwise stated) and were used without further purification. The alginates used in this study are listed in Table 5.

[0141] Table 5: Table of alginate formulations TIFF2024522379000032.tif37164

[0142] High-Throughput Modified Alginate Analogue Synthesis. A library of 211 next-generation analogues (including the critical triazole ring) was synthesized that incorporate further structural changes such as chain extension and differential substitution to generate high-resolution structure-function relationships.

[0143] A comprehensive strategy for the synthesis and characterization of alginates. An amide coupling reaction was carried out using 1 equivalent of UPVLVG alginate and 1 equivalent of an amine linker (five different amine linkers in Table 2) in the presence of 0.5 equivalents of a coupling agent of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride to obtain five different amine linker-conjugated alginate polymers. These linker-modified alginate derivatives were purified by dialysis (in saline and water) using a 10-12 kDa dialysis membrane for three days, followed by lyophilization. These five molecules were characterized for their purity and % modification of the starting alginate by NMR and elemental analysis. In the next step, one equivalent of an alkyne (45:alkyne) was coupled to the appropriately modified alginate by copper-catalyzed click reaction. A total of 211 triazole-containing alginate derivatives were obtained from the synthesis. All 211 different alginates were purified by dialysis followed by lyophilization and chemically characterized by NMR.

[0144] Optimized synthesis for small molecule preparation. Z2-A19 amine: 3-ethynylthiophene (1 eq, 4 g, 36.98 mmol) was added to a 1 L round bottom flask containing 420 mL of 5:1 methanol:water (350 mL of methanol and 70 mL of water) (5:1 methanol:water), followed by tris((1-benzyl-4-triazolyl)methyl)amine (0.25 eq, 2.932 g, 5.52 mmol) and stirred for 15 minutes. Triethylamine (0.25 eq, 0.77 mL, 5.52 mmol) and copper iodide (0.1 eq, 422 mg, 2.22 mmol) were then added. The reaction flask was cooled to 0° C. over 15 minutes while purging with argon, after which 11-azido-PEG-2-amine (1 eq, 5.86 mL, 36.98 mmol) was added. The reaction was stirred at room temperature for 5 minutes and then at 55° C. overnight. The reaction mixture was filtered through Celite® and the solvent was removed using a rotavap. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column using a mixture of dichloromethane:ultra (22% MeOH in DCM with 3% NH4OH) from 0% to 40%.

[0145] Z1-A3 amine: 1-Bromo-2-ethynylbenzene (1 equiv, 2 g, 11 mmol) was added to a 250 mL round bottom flask containing 180 mL of 5:1 methanol:water (150 mL of methanol and 30 mL of water) (5:1 methanol:water), followed by the dropwise addition of tris((1-benzyl-4-triazolyl)methyl)amine (0.25 equiv, 1.466 g, 2.76 mmol) dissolved in 24 mL of 5:1 methanol:water (20 mL of methanol and 4 mL of water) and stirred for 15 min., followed by the addition of triethylamine (0.25 equiv, 0.385 mL, 2.76 mmol) and copper iodide (0.1 equiv, 211 mg, 1.11 mmol). The reaction flask was cooled to 0° C. over 15 min while purging with argon, and then 11-azido-PEG-3-amine (1 eq, 2.193 mL, 11.05 mmol) was added. The reaction was stirred at room temperature for 5 min and then at 55° C. overnight. The reaction mixture was filtered through Celite® and the solvent was removed using a rotavap. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column with a dichloromethane:Ultra (22% MeOH in DCM with 3% NH4OH) mixture 0%-40%.

[0146] Synthesis of Z4-A10 amine: 1,3-Diethylbenzene (1 equiv, 4 g, 32 mmol) was added to a 1000 mL round bottom flask containing 450 mL of 5:1 methanol:water (375 mL of methanol and 75 mL of water), followed by dropwise addition of tris((1-benzyl-4-triazolyl)methyl)amine (0.25 equiv, 2.932 g, 5.52 mmol) dissolved in 30 mL of 5:1 methanol:water (25 mL of methanol and 5 mL of water) and stirred for 15 minutes. Triethylamine (0.25 equiv, 0.77 mL, 5.52 mmol) and copper iodide (0.1 equiv, 422 mg, 2.22 mmol) were then added. The reaction flask was cooled to 0° C. over 15 minutes while purging with argon, after which 11-azido-PEG-4-amine (1 equiv, 6.92 mL, 32 mmol) was added. The reaction was stirred at room temperature for 5 minutes and then at 55° C. overnight. The reaction mixture was filtered through Celite® and the solvent was removed using a rotavap. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column with a mixture of dichloromethane:ultra (22% MeOH in DCM with 3% NH4OH) 0%-40%.

[0147] Synthesis of B2-A17 amine: 3-iodobenzylamide (1 equiv., 4 g, 14.8 mmol) was added to a 50 mL round bottom flask containing 24 mL of methanol, followed by triethylamine (2.4 equiv., 3.6 g, 35.62 mmol), sodium azide (2 equiv., 1.93 g, 29.68 mmol), water (6 mL), copper iodide (0.15 equiv., 423.89 mg, 2.22 mmol), sodium ascorbate (0.1 equiv., 293.95 mg, 0.1 equiv., 2.97 mmol), and 1.83 mL of trans-N-N'-dimethylcyclohexene-1,2-diamine (0.2 equiv., 422.12 mg, 2.97 mmol). The mixture was evacuated and flushed with argon three times and (4-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)methanamine (1 equiv., 3.72 g, 14.8 mmol) was added. The reaction was stirred at room temperature for 5 min and then at 55° C. overnight. The reaction mixture was filtered through Celite® and the solvent was removed using a rotavap. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column with a dichloromethane:Ultra (22% MeOH in DCM with 3% NH4OH) mixture 0%-40%.

[0148] Synthesis of B1-A51 amine: (4-iodophenyl)methanamine (1 eq, 2 g, 15.13 mmol) was added to 90 mL of methanol. Triethylamine (2.4 eq, 3.67 g, 36.3 mmol) and sodium azide (2 eq, 1.97 g, 30.3 mmol) were added to the reaction flask. 40 mL of ultrapure water was added to the flask after all previous additions had dissolved. Sodium ascorbate (0.1 eq, 300 mg, 1.5 mmol) and copper iodide (0.15 eq, 432 mg, 2.27 mmol) were added to the flask. The flask was purged with argon by bubbling argon through the mixture for 15 min. Trans-N,N'-dimethylcyclohexane-1,2-diamine (0.2 equiv., 430 mg, 3.03 mmol) and 1-ethynyl-2-methoxybenzene (1 equiv., 2 g, 15.13 mmol) were added to the flask. The reaction was stirred at room temperature for 5 min, then at 55 °C overnight under argon. The reaction mixture was filtered through Celite® and the solvent was removed using a rotavap. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column with a dichloromethane:Ultra (22% MeOH in DCM with 3% NH4OH) mixture from 0% to 40%.

[0149] Synthesis of Z1-A34 amine: 4-propargylthiomorpholine 1,1-dioxide (1 eq) was added to a 250 mL round bottom flask and dissolved in a methanol:water mixture (5:1). To this was added tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methylamine (0.25 eq), triethylamine (0.25 eq), and copper iodide (0.1 eq). The reaction mixture was purged with argon for 15 minutes and cooled to 0° C. before adding 11-azido-3,6,9-trioxaundecan-1-amine (1 eq, 6.30 g, 28.86 mmol). The reaction mixture was stirred at room temperature for 15 minutes and then heated to 55° C. overnight. The reaction was cooled to room temperature and filtered through Celite® to remove any insoluble material. The filtrate was dried over silica using a rotavap under reduced pressure. The crude reaction was then purified by liquid chromatography on a 120 g ISCO silica column in a dichloromethane:ultra (22% MeOH in DCM with 3% NH4OH) mixture from 0% to 40% and further characterized by ESI and NMR mass spectrometry.

[0150] Alginate reaction: 1.5 g of VLVG (1 eq.) was dissolved in 45 ml of water. Then, 7.65 mmol of Z2-A19, Z1-A3 (1 eq.) amine was dissolved in 22.5 ml of acetonitrile and added to the mixture. Subsequently, an aqueous solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.75 eq.) was added dropwise to the mixture. The reaction was stirred at 55° C. overnight. The solvent was removed under reduced pressure and the solid was dissolved in water. The solution was filtered through a pad of cyano-functionalized silica and the water was removed under reduced pressure to concentrate the solution. It was then dialyzed against a 10,000 MWCO membrane in deionized water for 3 days. The water was removed under reduced pressure and lyophilized to obtain the functionalized alginate.

[0151] Synthesis of methacryloyl Z1A3 (Met-Z1A3): 2-(2-(2-(2-(4-(2-bromophenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethan-1-amine (1 equiv., 1.4 g, 3.51 mmol) was added to a 50 mL round-bottom flask and purged with argon three times (10 min each). 25 mL of anhydrous dichloromethane was added, followed by triethylamine (1.5 equiv., 734 μL, 5.265 mmol) and methacryloyl chloride (1.5 equiv., 509.6 μL, 5.265 mmol) dropwise under argon atmosphere. The reaction was stirred for 5-6 h and then purified on a 40 g ISCO silica column using 0%-40% dichloromethane:ultra (22% MeOH in DCM with 3% NH4OH) mixture to give approximately 1 g of a colorless solid (Met-Z1A3), which was characterized by mass spectrometry and NMR to confirm mass and purity.

[0152] Synthesis of methacryloyl B2-A17 (Met-B2-A17): (3-(4-(p-ridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)methanamine) (1.2 g, 4.78 mmol, B2-A17) was added to a 50 mL round bottom flask and purged with argon three times (10 min each). 25 mL of anhydrous dichloromethane (30 mL) was added, followed by the dropwise addition of triethylamine (1.5 equiv, 7.17 mmol, 999.6 μL) and methacryloyl chloride (1.5 equiv, 7.17 mmol, 693.99 μL) under argon atmosphere. The reaction was stirred for 5-6 h and then purified on a 40 g ISCO silica column using hexane / ethyl acetate (1:0 to 0:1) to give approximately 1 g of colorless solid (Met-B2-A17). The compound was characterized by mass spectrometry and NMR to confirm mass and purity.

[0153] Functionalization of alginates with small molecules: Functionalization of alginate with Z4-A10 amine: 1.5 g of UP-VLVG (1 eq.) was dissolved in 45 mL of water. Then, 7.65 mmol of Z4-A10 (1 eq.) amine was dissolved in 22.5 mL of acetonitrile and added to the mixture. Subsequently, an aqueous solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.75 eq.) was added dropwise to the mixture. The reaction was stirred overnight at 55° C. The solvent was removed under reduced pressure and the solid was dissolved in water. The solution was filtered through a pad of cyano-functionalized silica. The solution was then dialyzed against a 10,000 MWCO pre-treated dialysis tubing in deionized water for 3 days. The dialyzed solution was frozen at −80° C. and lyophilized to dryness.

[0154] Functionalization of alginate with B1-A51 amine: 1.5 g of UP-VLVG (1 eq.) was dissolved in 45 mL of water. Then, 7.65 mmol of B1-A51 (1 eq.) amine was dissolved in 22.5 mL of acetonitrile and added to the mixture. Subsequently, an aqueous solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.75 eq.) was added dropwise to the mixture. The reaction was stirred at 55° C. overnight. The solvent was removed under reduced pressure and the solid was dissolved in water. The solution was filtered through a pad of cyano-functionalized silica. The solution was then dialyzed against a pre-treated dialysis tube of 10,000 MWCO in deionized water for 3 days. The dialyzed solution was frozen at −80° C. and lyophilized to dryness.

[0155] Functionalization of alginate with Z1-A34 amine: 2 g (1 eq.) of UP-VLVG (BP-1903-04; Novamatrix) was dissolved in water (75 mL). Then, Z1-A34 small molecule (3.99 g, 10.20 mmol, 1 eq.) was dissolved in water under vortexing. The pH of the Z1-A34 aqueous solution was adjusted to 7.4 with HCl. Then, the Z1-A34 aqueous solution was slowly added to the UP-VLVG solution with stirring. Then, a solution of (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) (DMTMM, 0.5 eq.) was added dropwise to the mixture of UP-VLVG and Z1-A34. The reaction was heated to 55° C. and stirred overnight. The solution was filtered through a pad of cyano-functionalized silica. The solution was then dialyzed against 10,000 MWCO pretreated dialysis tubing in a beaker with saline (2 days) and Milli-Q water (3 days). The dialyzed solution was frozen at -80°C and lyophilized to dryness.

[0156] Gelation assay. 211 alginate analogs were tested in a fluorophore retention assay for their ability to efficiently crosslink to form hydrogels in the presence of barium. 100 μl of a 1% (w / v) solution of modified alginate polymer in 0.9% saline was dispensed into a 96-well plate. 1 μl of a 1% (w / v) solution of rhodamine B in DMSO was added to each well, followed by 50 μl of a 1 M barium chloride solution, then incubated for 10 min on an orbital shaker. Wells were washed three times with deionized water and then fluorescence was measured (ex: 540 nm / em: 580 nm). Previously synthesized UPVLVG alginate was used as a positive control and deionized water as a negative control.

[0157] Coating of catheters with Met-Z1-A3 / Met-B2-A17 using surface plasma cleaning. Barium-impregnated silicone rubber catheters (Codman® HOLTER® Atrial Distal catheter, Ref# 821670) were cut to the same length with a surgical blade. To perform chemical modification, the catheters were plasma treated with high frequency RF at a pressure of 600-700 mbar (Expanded Plasma Cleaner, Harrick Plasma, P / N PDC-001) three times at 1 min intervals, rotating to cover the entire surface between exposures, and immediately added to a 0.02 M solution of Met-Z1-A3 / Met-B2-A17 in 5% DMSO / toluene. The reaction was kept under stirring for 2 h and the implants were thoroughly washed three times in methanol, three times in ethanol, three times in sterile grade water, and finally again in sterile grade ethanol. Finally, the implants were vacuum dried overnight.

[0158] Scanning electron microscopy of coated and unmodified catheters Unmodified catheter samples were taped to SEM pin mounts, lightly treated with an air gun to remove particulates, and Au sputtered using a Denton Desk V Sputter system (Rice SEA). This was repeated for each group of coated catheters. The sputtered catheters were imaged using a JEOL 6500F scanning electron microscope.

[0159] XPS of unmodified and coated catheters X-ray Photoelectron Spectroscopy (XPS) is a surface-sensitive spectroscopic technique that quantitatively measures the elemental composition at the surface (within 6 nm range) of any material by irradiating a sample with monoenergetic X-rays, resulting in the emission of photoelectrons from the surface of the material. The elemental composition of uncoated, Met-Z1-A3 coated, and Met-B2-A17 coated implants was analyzed with a PHI Quantera XPS. The following inspection techniques were used for the analysis: 1100 eV, 200 μm spot size, 50 W 15 kV ion gun neutralization.

[0160] ToF-SIMS of unmodified and coated catheters Positive and negative high-resolution mass spectra were performed at the Shared Equipment Authority of Rice University using a ToF-SIMS NCS instrument combined with a TOF.SIMS5 instrument (ION-TOF GmbH, Muenster, Germany) and an in situ Scanning Probe Microscope (NanoScan, Switzerland). + Ion (measurement current 0.2 pA) was used as the primary probe, and the 250 × 250 μm 2 The field of view is 128 × 2128 pixel raster, 1.10 12 ions / cm 2 The static limit of the electron beam was taken into consideration to avoid damaging the surface. Charge compensation was performed using an electron flood gun during the analysis, and the surface potential was used to adjust for charge effects. The cycle time was fixed at 100 μs (corresponding to a mass range of m / z = 0 to 1102 a.mu).

[0161] Materials characterization and evaluation of catheters coated with lead molecules. XPS results showed that the small molecule-coated catheters had higher nitrogen content than the uncoated catheters, suggesting effective adhesion of nitrogen-containing small molecules to the catheters (Figure 14B). - The increased content supports the adhesion of small molecules to the catheter surface. The XPS results are further supported by Tof-SIMS results, which showed that the CN content was significantly higher in catheters coated with small molecules compared to uncoated ones. - The Br content was high in the catheter coated with bromine-containing Z1-A3. -The results showed that the Met-Z1-A3 and Met-B2-A17 catheters had higher urethane content (Figure 14C, Figure 15A, Figure 15B). In addition, the Met-Z1-A3 and Met-B2-A17 catheters appeared smoother than the unmodified catheters as observed from SEM images (Figure 15C), possibly due to the removal of microscopic ridges during the coating process.

[0162] Cell culture and proliferation. Human umbilical vein endothelial cells (HUVEC, CC-2517, LONZA, MD, USA) from 20 different donors (Table 3) were cultured in VascuLife® VEGF medium complete kit (LL-0003, Lifeline Cell Technology, CA, USA). HUVEC were subcultured for proliferation and maintained in a humidified incubator at 37°C with 5% CO2 atmosphere. The medium was changed three times weekly.

[0163] SNP profiles of 20 donors A 30-plex single nucleotide polymorphism (SNP) panel was designed to uniquely identify HUVEC donors by multiplex PCR reactions and next-generation sequencing (NGS). SNP loci were downloaded from the 1000 Genomes database, and genome sequences were obtained from the National Center for Biotechnology Information (NCBI) website. SNPs were selected to have population allele frequencies of 10% and 90% for the minor allele, and the loci were evenly distributed across chromosomes 1–22.

[0164] Genomic DNA (gDNA) was extracted from 20 different donors of HUVECs using the DNeasy kit (Qiagen, Cat#69054). For each donor, 100ng of gDNA was added to 50μL of PCR reaction mix containing 50nM of each primer and Phusion Hot Start Flex 2X Master Mix (NEB, Cat#M0536L). PCR reaction conditions included activation at 98°C for 30s, 20 cycles of denaturation at 98°C for 30s, annealing at 63°C for 2min, and extension at 72°C for 1min, followed by incubation at 72°C for 5min to complete the reaction (shortened to 98°C:30s-(98°C:10s-63°C:2min-72°C:1min)×20-72°C:5min-4°C hold). PCR products were purified using Monarch PCR & DNA Cleanup Kit (5 μg) (NEB, Cat#T1030S). The purified PCR products were then prepared for NGS on the Illumina Miseq platform using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Cat#E7645S) according to the manufacturer's protocol. Library quantification and quality control were performed on an Agilent 2100 Bioanalyzer. Deep sequencing was then performed at a depth of approximately 5000x to establish the SNP profiles of the 20 different HUVEC donors.

[0165] HUVEC encapsulation within modified alginate. Capsule preparation for mice. In each round of screening, 20 different materials were tested, and a total of 149 materials were screened in C57BL / 6J mice. Thus, for each round, 20 different HUVEC donors were used to generate cell barcodes corresponding to each material. The modified alginate was first dissolved in 0.8% saline at 3–5% w / v, and then mixed with 3% w / v SLG100 (also dissolved in 0.8% saline) in a volume ratio of 70% modified alginate to 30% SLG100. The alginate solution was sterilized by filtering through a 0.2 μm filter. Immediately before encapsulation, the cultured HUVECs were centrifuged at 250G for 5 min and washed with Ca-free Krebs buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 135 mM NaCl). After washing, the cells were centrifuged again and all the supernatant was aspirated. The cell pellet was then diluted with 5 × 10 6The donors were resuspended in alginate solution at a cell density of 100 μg / ml (approximately 40,000 cells / capsule). Each HUVEC donor was encapsulated with the corresponding modified alginate solution. Alginate capsules were made using an electrospray machine (Pump 11 Pico Plus, Harvard Apparatus, MA, USA). An 18G blunt needle was attached to a 1 ml Luer-lock syringe containing the alginate solution, which was fixed to a vertical syringe pump above a 150 ml crosslinking solution bath (20 mM BaCl2, 250 mM D-mannitol, 25 mM HEPES and 0.01 v / v% Tween20). A voltage generator was attached to the needle tip and grounded to the crosslinking bath. The syringe pump settings were a flow rate of 5 ml / hr at a height of 15–20 cm. The cell density per capsule was kept constant by adjusting the voltage between 5.5–7 kV. After the capsules were formed in the crosslinking bath, they were collected and then washed three times with HEPES buffer (25 mM HEPES (Gibco, Life Technologies, California, USA), 1.2 mM MgCl2×6H2O, 4.7 mM KCl, 132 mM NaCl), washed three times with culture medium, and cultured overnight in a 37°C incubator before implantation. Ten capsules of each material were dispensed into a 2 ml tube and mixed, and 200 capsules of this mixture were used for implantation. Just before implantation into the abdominal cavity of mice, the capsules were washed two more times with 0.9% saline. All materials were observed under a bright-field microscope to confirm uniform cell density and capsule size.

[0166] The top 10 lead materials identified from mouse screening and two controls (SLG20 and Z1-A34) were used for microcapsule implantation. Modified alginate was dissolved at 3-5% w / v in 0.8% saline and mixed with 3% w / v SLG100 in a 70:30 ratio. SLG20 was dissolved at 1.4% w / v in 0.8 saline. 300-500 μm sized capsules were made using the formulated alginate solution. The encapsulation procedure was the same as for 1.5 mm sized capsules, except that a flow rate of 200 μL / min and a 30G needle were used for microcapsules. After washing, 400 μL of microcapsules were dispensed and implanted into the IP cavity of mice for 2 weeks.

[0167] Creation of capsules for NHP implants: By mixing two different HUVEC donors in specific ratios, 400 different donor combinations can be created from 20 different donors to label biomaterials. To confirm the feasibility of mixed donor identification, different ratios (1:2, 1:3, and 1:4) were tested to determine the ratios detectable by NGS. A total of nine combinations were created using three cell lines (Figure 9A). After dispensing the correct ratios into the respective test tubes, capsules of nine different ratios were created using SLG20, followed by the encapsulation protocol described in the previous section. A total of 400 cell barcode combinations could be generated, including 20 single HUVEC donors, and 100 cell barcodes out of the 400 were used for NHP testing. A total of 100 cell barcodes were created, and these mixtures of HUVECs were encapsulated with 100 different materials each. Following the same encapsulation method as described in the previous section (mouse study), the cell density per capsule was fixed at 60,000 cells / capsule, which contained 20,000 from donor 1 and 40,000 from donor 2. Thirty capsules of each material were mixed in one T225 flask, and these mixed capsules were prepared for IP implantation. After dispensing the desired number of capsules into flasks, these samples were shipped overnight for implantation into NHPs.

[0168] Optimizing donor barcoding and identification A total of 400 donor pairs were generated as described above by combining two different donors from 20 donors. First, the mixing ratio of the two donors was optimized in vitro to test the feasibility of mixed donors. Three donor combinations (H15&H16, H16&H17, H15&H17) with three different mixing ratios (1:2, 1:3, 1:4) were used for this test, and subsequently, a total of nine different conditions were tested (Figure 9A). One capsule from each group was mixed and nine capsule donor pairs were analyzed (sample no. 1-9). Three different mixing ratios were tested to find the optimal condition with higher identification confidence (goodness level). Log-likelihood analysis was applied to deconvolute the donor identity. All samples were successfully identified with the matched donor pair. The goodness level decreased the more the cell densities of the two mixed cells moved away from each other. The mixing ratio of donor 1:donor 2 was set at 1:2 as the optimal condition for further experiments (Figures 9B-9D).

[0169] Next, the identifiability of three different materials was tested in mice under the in vitro optimized conditions. Three materials (Z1-A34: positive control that reduces FBR, PVLVG: unmodified control, and B1-A51: negative control that cannot reduce fibrosis) were prepared to encapsulate two donor pairs in a 1:2 ratio. A mixture of the three materials (20 capsules / material in each mouse) was implanted into the IP cavity of mice (M1-M3) for 2 weeks (Figure 10A and Figure 10B). After the capsules were retrieved (Figure 10C), they were divided into three groups based on the fibrosis level (Figure 10D). A total of 45 capsules were selected for donor identification and the corresponding materials were determined (Figure 10E and Figure 10F). Of the 400 donor combinations, 43 / 45 (95.6%) donor pairs were successfully identified, suggesting that this mixed donor barcoding strategy could be applied to screen hundreds of materials in larger animal models. Results from this cohort showed that Z1-A34 (an immune-protective material), as previously reported, had the highest number of low fibrotic capsules, followed by UP-VLVG (unmodified alginate). The ability to screen hundreds of materials with this dual barcoding strategy will exponentially expand the high-throughput potential of screening to identify materials that mitigate FBR.

[0170] Human islet encapsulation with lead material: Human islets (Prodo Labs) were cultured in PIM(S) medium (Prodo Labs) for further use. The cultured islets were centrifuged at 1200 rpm for 3 min and washed with Ca-free Krebs buffer. The islets were then centrifuged again. The islet pellet was then resuspended in a 5% solution of Z4-A10 (mixed with 3% SLG100 in a 70:30 ratio) at an islet density of 5,000 islets / 1 mL alginate solution. The capsules were cross-linked in BaCl2 solution and adjusted in size to 1.5 mm. After cross-linking, the capsules were washed three times with HEPES and re-washed twice with medium. As islets have a variety of sizes (50-400 μm), the total number of encapsulated islets was re-counted and converted to islet equivalents (IEQ) (Figure 12). The average IEQ for each capsule was approximately 10 IEQ / capsule (1x). To make high density capsules (2x and 4x density), the amount of alginate was reduced to 0.5mL and 0.25mL, respectively, while maintaining the same IEQ. Finally, Z4-A10 modified alginate produced approximately 20 IEQ / capsule (2x) and approximately 40 IEQ / capsule (4x). SLG20 was used as a control material, followed by the same encapsulation method and islet density.

[0171] Optimization of DNA extraction (in vitro): To increase the amount of gDNA extracted from one capsule, different parameters were optimized using the DNesay kit (Qiagen, catalog #69054). Fresh and flash-frozen capsules were compared with and without lysis conditions. 50 mM EDTA in 10 mM HEPES solution was applied for capsule lysis, followed by centrifugation at 250G for 5 min. Only pelleted cells were used for the subsequent DNA extraction step. Undissolved capsules were also used after homogenization as a comparison. Then, the yield was tested for different elution temperature settings (RT vs. 56°C). Finally, different cell numbers per capsule (5,000, 10,000, 20,000, 40,000, and 80,000 cells / capsule) were compared to determine the optimal cell density for in vivo testing.

[0172] Optimization of DNA extraction and NGS identification from single capsules Because each individual hydrogel capsule contains a unique genotype from a different HUVEC, extracting gDNA from each capsule is an essential first step to identify the barcode of each material.

[0173] The cells reside within the cross-linked hydrogel matrix, which makes it difficult to separate the cells from the hydrogel. The cell separation and gDNA extraction steps were optimized to increase the gDNA content by comparing the lysis composition, cell density, and extraction temperature (Figure 4). Using pre-implanted HUVEC capsules, the DNA extraction efficiency under different conditions was compared. First, each capsule was dissolved with EDTA solution. Dissolving the capsules with EDTA before extracting DNA from one capsule improved the DNA extraction efficiency by about five-fold (Figure 4A). It was also confirmed that the quick-frozen capsules showed similar DNA content as the fresh samples, and the excised capsules could be stored for future use after the quick-freezing step. In addition, to increase the amount of extracted DNA, different elution conditions were compared (Figure 4B). When the samples were eluted with pre-warmed elution buffer at 56 °C, the DNA content increased. In addition, the total DNA amount also increased as the elution volume increased. However, the DNA concentration decreased with increasing elution volume, and 100 μl of elution buffer was selected as the optimal volume for further analysis. Finally, the number of cells per capsule was adjusted by comparing different cell density conditions (Figure 4C). With increasing cell density per capsule, the extracted DNA content increased. However, the total DNA yield decreased, therefore, approximately 20,000 cells / capsule was set as the minimum cell density for encapsulation. The optimized conditions were applied to extract gDNA from excised capsules, and these extracted gDNA samples were used for optimizing the NGS method.

[0174] We successfully extracted DNA with high quality and sufficient human DNA content from in vitro and in vivo excised capsules for polymerase chain reaction (PCR) amplification and subsequent NGS for SNP genotyping. Furthermore, the NGS library preparation was optimized to increase the on-target rate even at low DNA input (Figure 5). Finally, a bioinformatics pipeline illustrates a high-throughput strategy of the NGS analysis process for material / donor identification (Figure 6).

[0175] Optimization of NGS library preparation workflow: Because DNA extracted from capsules was at low concentration and the input amount for NGS library construction was typically less than 1 ng, PCR reactions were highly prone to primer dimers, especially in multiplex PCR. Here, we optimized the library preparation workflow to reduce primer dimers. The first PCR amplified SNPs with multiplex primers containing 5'-overhang sequences. The primer concentration, PCR cycles, and annealing time of the first PCR were adjusted to reduce primer dimers that may result from multiplex PCR. After on-plate purification, the second PCR modified the position barcodes by amplifying with row-specific and column-specific primers containing Hamming barcode sequences and sequences that anneal to the 5'-overhang regions of the SNP primers. The amplification cycles of the second PCR were also adjusted.

[0176] Implant / implant surgery. IP implantation of mixed capsules in C57BL / 6J mice: All mouse experiments were approved by Rice University's Institution Animal Care and Use Committee (IACUC). Immunocompetent male C57BL / 6J mice were first weighed and anesthetized with 1–4% isoflurane in oxygen on a heating pad. Buprenorphine was administered subcutaneously based on body weight (0.5 mg / kg dose). The abdomen was shaved and disinfected with betadine and isopropanol scrubs, three times each. A 0.5–10 cm midline incision was made in the skin with a sharp blade. The peritoneal wall was then grasped with forceps and a 5 mm incision was made along the linea alba. A 0.5 ml volume of capsules was then implanted in the abdominal cavity. The abdominal muscles were closed with absorbable sutures. The skin was closed with sutures.

[0177] Subcutaneous implantation of catheter samples in C57BL / 6 mice: Unmodified and coated catheters were implanted into the subcutaneous space of n=6 C57BL / 6 mice (Charles River Labs). Specifically, one incision site was made on the back of each mouse, and a separate subcutaneous pocket was created for each catheter implantation. The incisions were sutured closed, and the mice were monitored and cared for according to the standards of the Rice Animal Resource Facility.

[0178] IP transplantation of human islet capsules and blood glucose monitoring in STZ-induced diabetic C57BL / 6J mice: To generate insulin-dependent diabetic mice, healthy C57BL / 6J mice were treated with streptozotocin (STZ). STZ solution at a concentration of 7.5 mg / ml (50 mg / kg of STZ) was injected into the IP cavity for 5 consecutive days. After fasting for 1 h, blood glucose (BG) levels and body weights of all mice were measured. Only mice with BG levels above 350 mg / dL for 2 consecutive days were considered diabetic and used for islet transplantation. In the 1× group, 200 capsules containing human islets were transplanted into diabetic mice (approximately 2,000 IEQ per mouse). Additionally, 100 capsules at approximately 20 IEQ / capsule and 50 capsules at approximately 40 IEQ / capsule were implanted in the 2x and 4x groups, maintaining the same number of IEQ per mouse (approximately 2,000 IEQ per mouse). After transplantation of islets containing Z4-A10 and SLG20 capsules, BG levels were monitored three times a week. Mice with BG levels below 250 mg / dL were considered normoglycemic. Monitoring continued until all mice had returned to a hyperglycemic state, at which point they were euthanized and capsules were retrieved. Mice were fasted for 4 hours before in vivo glucose tolerance testing. Each mouse received a bolus of 1.5 g / kg of 30% sterile glucose solution in saline via tail vein injection. Blood glucose levels were measured every 15 minutes for 2 hours after glucose injection.

[0179] Laparoscopic capsule implantation into the IP cavity of NHPs: On the scheduled day of surgery, NHPs (male Mauritian cynomolgus monkeys) were sedated and anesthetized (according to an approved animal protocol). The anterior abdomen was shaved and prepared from the xiphoid process to the pubis. A small (2 cm) supraumbilical incision was made and a 5-12 mm trocar was inserted. Pneumoperitoneum was created with CO2 at a pressure of 10-14 mmHg. After warming with warm saline, a camera was inserted into the abdominal cavity through the trocar. Under laparoscopy, another two small incisions (1-2 cm) were made (left and right flanks) and a 5-12 mm trocar was inserted into the abdominal cavity. A 2 mL sterile pipette connected to a syringe by a silicone tube was inserted into the abdominal cavity through the trocar. The capsule was evenly distributed around the liver, retrogastric, perisplenic, left and colonflexium, omentum, and retrosmall intestine. The three small incisions were then sutured closed in layers with Vicryl 3-0 for the muscle and Vicryl 4-0 for the skin (subcutaneous). The animals' recovery was observed by operating room staff, veterinary staff, and technicians. The animal protocol, including the care and use of non-human primates in this study, was reviewed and approved by the University of Illinois-Chicago (UIC) Institutional Animal Care and Use Committee (IACUC) prior to initiation. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of UIC.

[0180] Recovery of materials. Capsule recovery from the IP cavity: At a specific time of implantation, five mice from each round were euthanized under CO2 administration followed by cervical dislocation. An incision was then made along the abdominal skin and peritoneal wall using forceps and scissors. Then, all material capsules were washed from the abdomen using Ca+ Krebs buffer and collected in a Petri dish. After ensuring that all capsules were washed or manually collected, they were transferred to a 50 ml conical tube. After several washes with Krebs buffer, the mixed excised capsules were processed for further imaging and selection.

[0181] Capsule retrieval from the IP cavity of NHPs using laparoscopic technique: NHPs were sedated and anesthetized (according to approved protocol). The anterior abdomen was shaved and prepared from the xiphoid process to the pubic bone. A 2 cm supraumbilical incision was made and a 5-12 mm trocar was inserted. Pneumoperitoneum was created with CO2 at a pressure of 10-14 mmHg. After warming with warm saline, a camera was inserted into the abdominal cavity through the trocar. Under laparoscopy, another incision (1-2 cm) was made (left and right flanks) and a 5-12 mm trocar was inserted into the abdominal cavity. Laparoscopic images and videos were taken to document the distribution of microcapsules. 20-30 cc of saline was used to laparoscopically wash out any loose capsules that may have been present in the pelvic cavity that may have leaked from the omental pouch. The animal protocol, including the care and use of non-human primates in this study, was reviewed and approved by the University of Illinois-Chicago (UIC) Institutional Animal Care and Use Committee (IACUC) prior to initiation. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of UIC.

[0182] Processing, fixation, and histological examination of catheter explants: Catheters were explanted at 4 weeks by carefully removing the catheter and attached tissue together. The subcutaneous catheter explants were covered with a thin membrane that was strongly attached to the skin and lightly attached to the muscle (Figure 15). The tissue around the catheter was cut approximately 3 mm from the catheter, and the catheter with the skin attached was removed from the mouse. The explants were fixed in 10% formalin (Sigma) for 4 days and then transferred to PBS. Further processing, sectioning, and histological examination were performed by the Baylor Pathology and Histology Core. Specifically, the samples were embedded in paraffin, cut along the cross-sectional axis of the catheter, and H&E stained.

[0183] Imaging and selection of excised material capsules: For phase contrast imaging, the capsules were gently washed with Krebs buffer, transferred to a 35 mm Petri dish, and bright-field and dark-field imaging was performed using a Leica microscope. Based on the level of fibrotic hyperplasia on the capsule surface, the capsules were manually divided into three different groups (L: low fibrosis group, M: medium fibrosis group, and H: high fibrosis group). The selected transparent capsules with less fibrosis (L group) were snap frozen and used for DNA extraction, while the rest were snap frozen in liquid N2 and stored at -80°C for further use.

[0184] Live / Dead Cell Staining: Fluorescent imaging of cells stained with the Live / Dead assay was performed to examine the viability of encapsulated HUVECs from capsules either pre- or post-implantation. Five capsules of each material were washed with DPBS and stained with 2 μM calcein AM and 4 μM EthD-1 in complete medium. Capsules were incubated for 30 min and imaged using an EVOS microscope with fluorescence filters. Live cells were imaged green with a GFP filter and dead cells red with a Texas-Red filter. (Excised Capsules-NSG Mice) For capsules excised from NSG mice, capsules were washed three times with Ca+ Krebs buffer and then incubated in staining solution. Capsules from each mouse were transferred to a 35 mm Petri dish and washed twice with DPBS. Images were taken at 2x magnification and the resulting images were stitched together to view the entire Petri dish.

[0185] Dithizone staining: The isolated islet capsules were stained with dithizone (DTZ). 5 mg of DTZ was dissolved in 1 mL of dimethyl sulfoxide (DMSO), mixed thoroughly, and incubated for 5 min. 4 mL of DPBS was added to the mixed solution and filtered through a 0.22 μm filter. The islet capsules were placed in a 35 mm Petri dish and washed three times with PBS. The capsules were then incubated in the DTZ solution for 5 min and washed three times with DPBS to remove background staining. The stained capsules were imaged with a Leica microscope.

[0186] Protein extraction from the recovered microcapsules and quantification of collagen content: Cells / tissues deposited on the microcapsule surface were lysed using RIPA buffer (catalog #89901, Thermo Scientific, PA, USA) for protein extraction. Briefly, a ratio of 100 μl microcapsules to 200 μl lysis buffer containing Halt™ Protease Inhibitor Cocktail (catalog #78430, Thermo Scientific, PA, USA) was used for cell lysis from capsules. The lysate was centrifuged at 12000 rpm for 20 min at 4°C and the supernatant was transferred to a new tube. The pellet was washed with the same volume of lysis buffer and then centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was combined with the previous one and the extracted protein was stored at -80°C for future use. Protein concentration in the lysate was quantified using a BCA assay (Pierce BCA Protein Assay Kit, catalog #23225, Thermo Scientific, PA, USA). Lysates from each sample containing 20 μg of protein were diluted to 100 μL with water, mixed with 37% hydrochloric acid in a 1:1 ratio, and then hydrolyzed at 120 °C for 3 h. The resulting solutions were used to measure the collagen content of the recovered microcapsules using a Hydroxyproline Assay Kit (Cat. #MAK008, Sigma-Aldrich, MO, USA) according to the manufacturer's instructions. Absorbance at 560 nm was measured and the blank value of the hydroxyproline standard was subtracted from all measurements. Hydroxyproline content was determined from a hydroxyproline standard curve.

[0187] Immunofluorescence staining for confocal imaging: For immunofluorescence staining, the collected microcapsules were washed with Krebs buffer and fixed in 4% paraformaldehyde overnight at 4° C. Samples were washed three times with PBS and cells were permeabilized with 1% Triton X-100 for 15 min at room temperature. After washing with PBS, the samples were incubated in 1% bovine serum albumin (BSA) solution for blocking at room temperature for 1 h, and then incubated with staining solution containing antibody cocktail (Alexa Fluor 488 anti-mouse CD68 antibody (catalog #137012, BioLegend, CA, USA) at a dilution of 1:200 in 1% BSA, Anti-mouse α-Smooth Muscle-Cy3 (catalog #C6198, Sigma-Aldrich, MO, USA) at 1:200, and 2 drops / ml DAPI (NucBlue Fixed Cell ReadyProbes Reagent, catalog #R37606, Invitrogen, CA, USA) at room temperature for 1 h. After washing with 0.1% tween20 solution, the samples were washed twice with PBS and transferred to 50% glycerol solution in glass-bottom 24-well plates for imaging. A Nikon A1-Rsi confocal microscope was used for immunofluorescence imaging.

[0188] Histological processing of retrieved catheters (H&E and Masson's trichrome staining) and histological evaluation of tissue hyperplasia on the catheters: Stained sections of catheter explants were imaged with a Leica M165C light microscope. To determine the extent of tissue hyperplasia on the catheters, the dark zone of purple tissue between the catheter and skin layer was measured using ImageJ software. This method is based on the findings of Xie et al. (2018), who showed that the tissue zone directly adjacent to the subcutaneous implant stains dark purple with H&E and blue with trichrome. The blue staining with trichrome means that this tissue zone is rich in collagen, thus suggesting that this tissue exhibits fibrous hyperplasia. Because of this correlation, the dark purple tissue was measured and used to evaluate the relative immune response to the differently coated catheters.

[0189] Specifically, images of catheter sections were rotated so that the adjacent skin tissue was facing down. For each image, the dark purple band of tissue was measured three times at equal intervals along the skin tissue and averaged to avoid arbitrarily measuring one spot. ImageJ pixel measurements were converted to millimeters by measuring the length of a scale bar burned into the image. Note that some sections have larger, light purple bands of tissue that run more perpendicular to the catheter. These were specifically avoided during measurements as they are part of the scar tissue from the incision.

[0190] DNA extraction from excised capsules and RT-qPCR. For DNA extraction from single capsules, the method optimized for this study was applied. Encapsulated cells from pre- or post-excised capsules were lysed in 50 mM EDTA for 15 min and centrifuged at 5000 rpm for 10 min. The supernatant was aspirated and the cell pellet was suspended in 200 μl of PBS. Total gDNA was isolated from single capsules using the DNeasy kit (Qiagen, Cat. No. 69504) according to the manufacturer's instructions with slight modifications and optimized conditions. Briefly, the cell suspension was lysed with proteinase K and RNase A for 5 min at RT and incubated with lysis buffer at 56 °C for 20 min. After addition of ethanol, the supernatant was transferred to a column and washed twice with wash buffer. DNA was recovered with elution buffer heated at 56 °C and stored at -20 °C for further use. Total RNA was extracted from 100 μl of recovered microcapsules (300-400 μm size). Quick-frozen capsules were thawed on ice, homogenized, and processed using the RNeasy Mini Kit (Qiagen, Cat. No. 74104) according to the manufacturer's instructions. Extracted RNA was converted to cDNA for RT-qPCR using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4368814). Real-time qPCR was performed with 3 μL of gDNA / cDNA in a 10 μL reaction volume with SYBR Green (PowerUp SYBR Green Master Mix; Applied Biosystems, Cat. No. A25741) to quantify PCR products. PCR was performed under the following conditions: 95°C for 10 s, 48°C for 20 s, 72°C for 30 s (40 cycles), 72°C for 5 min, 65°C for 5 s, and a final cycle at 95°C. All reactions were performed in triplicate. Data were analyzed by the 2-ΔΔCT method and relative RNA levels were compared after normalization to mouse b-actin (ActB) and SLG20 (control). Primers used in this study are shown in Table 6.

[0191] Table 6: List of RT-qPCR primers TIFF2024522379000033.tif45158

[0192] Next generation sequencing to identify low-fibrotic, immunomodulatory alginate analogs. NGS Library Preparation. DNA content in individual capsules was semi-quantitatively assessed using qPCR as a quality control procedure for the samples, with Ct values ​​negatively correlated to extracted DNA content. Samples with amplifiable DNA content underwent two PCR steps to amplify and barcode the target amplicons. With DNA from each capsule in individual wells of a 96-well PCR plate, the first PCR was performed using 30-plex primers targeting 30 non-pathogenic SNPs whose genotype profile uniquely identifies the HUVEC donor (Table 7). All 30-plex primers contained 5'-overhang sequences and incorporated a universal binding domain for the target amplicon. The reaction mixture consisted of 30-plex SNP primers at a concentration of 50 nM each and 1X of Phusion Hot Start Flex 2X Master Mix. The reaction conditions were activation at 98°C for 30 seconds, and 7 cycles of denaturation at 98°C for 30 seconds, annealing at 63°C for 5 minutes, and extension at 72°C for 1 minute, with a 5-minute incubation at 72°C to terminate the reaction (shortened to 98°C:30 seconds-(98°C:10 seconds-63°C:5 minutes-72°C:1 minute) x 7-72°C:5 minutes-4°C:hold). AMPure XP magnetic beads (Beckman Coulter, Cat. No. A63881) are added to the first PCR product in a 1.2-fold volume ratio. The suspension is incubated at room temperature for 5 minutes, then placed on a magnetic stand, and the supernatant is separated and discarded. The remaining magnetic beads are washed twice with 80% ethanol, and the DNA contents are eluted in water. In the second PCR, the capsule samples were uniquely barcoded using primers with overhanging Hamming code sequences. A total of 12 barcoded column-specific forward primers and 8 barcoded row-specific reverse primers were designed with a distance of at least 3 from each other, allowing barcoding of 12 x 8 = 96 capsules. The reaction conditions were 98°C: 30 sec-(98°C: 10 sec-63°C: 1 min-72°C: 1 min) x 20-72°C: 5 min-4°C: hold), primer concentration was 400 nM, and Phusion Hot Start Flex 2X Master Mix was 1X.The row and column barcodes uniquely determined the location of the samples on the 96-well plate, therefore barcoded amplicons could be pooled from all wells to form a single library containing SNP information from 96 different capsule samples (Table 8). Products were purified using AMPure XP magnetic beads at a volumetric ratio of 1.0× as previously described.

[0193] Illumina sequencing adapters were modified using a ligation-based method, and library indexes were added by PCR using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Cat. No. E7645S) according to the manufacturer's instructions. Library quantification and quality control were performed on an Agilent 2100 Bioanalyzer.

[0194] Table 7: SNP information and SNP primer sequences. TIFF2024522379000034.tif133153TIFF2024522379000035.tif244153TIFF2024522379000036.tif41153

[0195] Table 8: Position barcoding primer sequences TIFF2024522379000037.tif121161

[0196] Analysis of single donor samples. Encapsulated materials were barcoded by co-encapsulated HUVEC donor cells, and thus the identity of the HUVEC donor could be determined by sequencing data analysis to reveal material information. NGS fastq data were demultiplexed by row and column barcodes to realign sequences amplified from the same DNA input. Sequence alignment to the target amplicons was performed with bowtie2. For each amplicon sequence, the grep function was then applied to search for dominant and mutant alleles, and the mutant allele frequency (VAF) of each SNP locus was calculated. Because the capsules implanted in mice contained only one HUVEC donor, the HUVEC donor with the highest concordance with the analyte sample was identified as the barcoding cell.

[0197] Analysis of dual donor samples: We analyzed explant samples encapsulating one or two HUVEC donors using log-likelihood. Specifically, we calculated SNP VAF profiles for all possible compositions of donor cells. The VAF in Eq. (1) i,j,k represents the expected VAF of the kth SNP when donor i and donor j are mixed in a ratio of 1:R, where all possible combinations of 20 different HUVEC donors had 20 × 20 = 400 different compositions. For each possible composition, the observed VAF of each SNP was calculated from a Gaussian distribution with a probability, p(ij,k), depending on how close or far the observed value was from the VAF of the composition. The overall log-likelihood of each composition, Log(L i,j ) is obtained by summing the log-likelihoods of all SNPs, and the composition with the highest overall log-likelihood is determined as the barcoding cell composition. TIFF2024522379000038.tif25128

[0198] Statistical analysis. Statistical analysis of material screening was performed using customized MATLAB scripts. Recovery rates of good materials were averaged among biological replicates (n=5 mice). Error bars with 95% confidence intervals were calculated for each material from binomial distribution. Any two materials with non-overlapping error bars were identified as statistically significant (p<0.05). Statistical analysis of other graphs used one-way or two-way ANOVA with Bonferroni multiple comparison correction (****P<0.0001, ***P<0.002).

[0199] C. Results and Discussion Material and Method Development for NGS Screening and Screening in Immunocompromised Mice. To screen a large library of immune-protective hydrogels in vivo using cell barcoding, a sequential method for identifying cell encapsulating materials followed by in vivo screening of cell encapsulating materials was developed, comprising: (a) preparing a plurality of barcoded cells from one or more subjects, each composition of barcoded cells containing a unique profile of SNPs that serve as a genetic barcode for each cell encapsulating material; (b) creating capsules using various encapsulating materials and barcoded cells; (c) implanting the capsules in vivo in test subjects; (d) extracting the capsules after a set period of time; and (d) sequencing the SNPs in the barcoded cells of each extracted capsule, thereby identifying the cell encapsulating material of each capsule (FIG. 2A). A previously published report demonstrated the development of a 774 combinatorially synthesized hydrogel library and the identification of three lead triazole-containing antifibrotic covalently modified alginate analogs with similar molecular structures (1). Careful structural analysis showed that the commonality among the lead analogs was the presence of a triazole (a heterocyclic five-membered ring with two carbon and three nitrogen atoms). This suggests that triazole-containing molecules may modulate immune cell populations on these biomaterial surfaces, including macrophages, inhibiting their activation and interfering with the fibrotic process. In this manuscript, a total of 211 new alginate analogs (encapsulating materials) were synthesized by triazole surface modification (Figure 1). By keeping the triazole analogs common across all, we developed a combinatorial biomaterial approach to generate a library of alginate-based hydrogels with covalently attached small molecule functional groups. Ultrapure alginate UPVLVG (>60% G, approx. 25 kDa MW, NovaMatrix) with low molecular weight (MW) and high guluronic acid (G) content was used as the starting material. Three hydrophilic PEG-based linkers (Figure 1, Table 2) were used to generate 150 unique polymers.Another 61 unique polymers, all containing triazoles, were generated using two hydrophobic linkers (Figure 1, Table 2). Furthermore, the covalent conjugates were characterized using elemental analysis, nuclear magnetic resonance spectroscopy (NMR), and gelation assays (Figures 3D, 3E, and 3A, respectively). After initial characterization tests including purity, solubility, and gel-forming ability, about 149 alginate analogs were selected for in vivo screening (Table 3). Furthermore, various in vitro characterizations of the newly formed capsules with or without cells were performed using 1) live / dead assays using fluorescence-based imaging of human umbilical vein endothelial cells (HUVECs), and 2) bright-field and dark-field microscopy to verify the homogenous shape and size of all capsules. 20 unique donors of HUVECs were sequenced by next-generation sequencing (NGS) and unique barcoding was established to identify their individual single nucleotide polymorphisms (SNPs), which were used as barcodes to tag and identify the different encapsulated materials. DNA extraction was successfully performed from in vitro and in vivo excised capsules, yielding high-quality and sufficient human DNA for polymerase chain reaction (PCR) amplification and subsequent NGS for human SNP identification. Deep sequencing of HUVEC cells revealed the genetic profile of the selected 30 non-pathogenic SNPs. Each SNP locus could have one of three genotypes: homozygous for the wild-type (WT) allele, heterozygous, or homozygous for the mutant allele. Collectively, the genotypes of the 30 SNPs formed a unique genetic profile that could obscure HUVEC cell identity (Figure 2B). Figure 2C is a representative image from the screening (pre- and post-implantation). Thus, by encapsulating a single donor HUVEC in each distinct hydrogel material, the identity of the material is uniquely tagged by a cellular barcode that can be read by sequencing after implantation. We optimized the gDNA extraction method from a single capsule (Figure 4) to increase the DNA input for sequencing. Furthermore, we established a workflow for NGS library preparation and material identification (Figure 5 and Figure 6).

[0200] To show that all donors (H1-H20, Table 3) could be deconvoluted by NGS, a mixture of 20 different hydrogel capsules (made from a library of 211 novel hydrogel analogs) encapsulating 20 unique HUVECs was implanted intraperitoneally (IP) into NSG (NOD SCID gamma) mice for 4 weeks. NSG mice lack mature T cells, B cells, and natural killer cells to induce normal innate immune function. Screening in immunodeficient mice allows the inventors to evaluate this cell barcoding strategy in conditions with minimal host immune response to the implanted material. Imaging of the capsules after retrieval showed minimal cell deposition, no fibrosis, and high viability of the encapsulated cells (Figure 2C). Capsules from one mouse were analyzed by NGS technology, and 195 of 200 capsules (~96.5%) were successfully identified based on their unique cell barcoding (Figure 2D). The identified percentage was evenly distributed among the donors. These results indicate that cell barcoding using different HUVEC donors and NGS genotyping can be utilized as a suitable strategy to determine biomaterial identity without altering the material properties.

[0201] Material screening in immune-competent mice. To measure the level of immune response, these newly synthesized alginate analogs were evaluated using a novel high-throughput in vivo screening method (Figure 7A). Each material was barcoded using the unique single nucleotide polymorphism (SNP) genotypes of 20 different HUVEC donors. A mixture of 20 different materials (10 capsules for each material) was implanted intraperitoneally (IP) in each mouse for each round. A total of approximately 150 novel materials were screened using this method. 28 days after in vivo implantation in mice, capsules were retrieved from the IP cavity and post-processed to identify the donor pair of materials (Figure 7B). Capsules showing a low fibrotic response suggest, without being bound by theory, that the materials have immune-protective properties. Since these excised capsules were a mixture of 20 different materials, the encapsulated HUVEC donors should be identified using a high-throughput method to find the corresponding materials. All excised capsules were classified into three groups (L: low, M: medium, H: high fibrosis capsules) based on the surface fibrosis level under a microscope. Capsules in the low fibrosis (L) group had less fibrosis deposition and a clear surface. In contrast, the high fibrosis (H) group had more deposition of fibrotic tissue on the capsule surface, resulting in the formation of aggregates due to fibrotic hyperplasia between capsules. Capsules between the two groups were assigned to the moderate fibrosis group (M). Only capsules in the L group were selected for material identification using NGS assay (Figure 7C).

[0202] To increase the throughput of material screening, we designed a workflow to process a batch of 96 extracted DNA samples at a time. After amplifying SNP amplicons in a 96-well PCR plate, each sample on the plate is positionally barcoded by a unique combination of forward and reverse primers containing a Hamming code that represents their position on the plate. This allows the barcoded amplicons to be pooled without losing sample information, and the pooled library can then be processed for sequencing with a ligation-based method on the Illumina platform. For in vivo screening in mice, two such libraries could be screened per capsule excised from one animal, with an estimated sequencing space of approximately 11.5 million reads (based on 2000 coverage / plex × 30 plex × 96 samples / library), or 2.9% of the capacity of a NextSeq flow cell.

[0203] All identified capsules were plotted (Figure 7E) to find hit materials with anti-inflammatory properties. The percentage of low fibrotic capsules represented the anti-fibrotic performance of the materials (Figure 7E, Table 3). In each round, 10 capsules were implanted per material, and thus the graph was plotted as the percentage of low fibrotic capsules (compared to the number of implanted capsules). As a result, a total of 15 alginate analogs (blue and orange bars in Figure 7E) (including one known material, Z1-A34, shown in green bars in Figure 7E) were found to exhibit low fibrotic capsules of more than 50%, which means that on average 5 capsules out of 10 implanted capsules were transparent. In addition, to visualize the fibrotic results of different combinations of alkynes and linkers, the screening results were plotted as a heat map (Figure 7D). From these results, it is clear that some of the alkynes (A3, A17, A19, A30, A43) exhibited low fibrotic levels with different linkers. These alkynes contain phenylbromine, pyridine, thiophene, ethoxy, and cyclopropyl functional groups, respectively (Table 2). Some similarities among these alkynes include the lack of branched structures and long carbon chains, making these alkynes relatively compact. In addition, these alkynes all contain carbon ring structures, most of which have electronegative atoms (O, N, S, Br) in or around the carbon ring.

[0204] Figure 7F shows the chemical structures of the top three lead alginate analogs we identified (Z4-A10, Z2-A19, and Z1-A3) with the lowest FBR or fibrosis (orange bars in Figure 7E). All three new analogs contain a triazole moiety in the backbone, further supporting the role of the triazole in hindering fibrosis. Interestingly, all three lead materials have a hydrophilic PEG linker attached to the alginate in addition to the bromobenzene (Z1-A3), thiophene (Z2-A19) and ethynylbenzene (Z4-A10) functional groups on the triazole (Figure 7F). Notably, among the top 15 leads, only two hydrophobic analogs were identified (B2-A17 and B1-A34), one of which was later used for catheter coating application.

[0205] Dual donor cell barcoding expands the potential for high throughput. To translate this novel barcoding strategy to screen larger batches of antifibrotic biomaterials, we utilized a dual donor barcoding strategy and tested its feasibility in the NHP model. Only 20 different materials were implanted in one mouse. In contrast, the larger volume of implantable space in the NHP model allowed us to increase the batch size of materials to 100, resulting in a 5-fold increase in throughput. In single donor encapsulation, the throughput is limited by the number of unique donors available. Here, we devised a new method using a dual donor barcoding strategy to increase the throughput of screening without purchasing and validating new cell donors. By mixing two different HUVECs in a 1:2 ratio (Figure 8A), we generated 400 unique barcoding composition permutations with only 20 HUVEC donors (Figure 8B), which significantly expanded the barcoding capacity of existing donors. With this ratio, the convoluted variant allele frequency (VAF) contains seven possibilities (0, 1 / 6, 1 / 3, 1 / 2, 2 / 3, 5 / 6, and 1). Therefore, we change the donor identification strategy from a direct comparison with individual donors to a log-likelihood analysis (Figure 6). The likelihood of all possible combinations was evaluated from a Gaussian distribution to determine the identity of the mixed donor. The mixing ratio of the two donors was optimized (Figure 9), and a small cohort screening (comparison of three materials) was performed to confirm the feasibility of the dual donor barcoding strategy (Figure 10).

[0206] After development of 400 novel dual barcodes, a set of 100 unique triazole-containing alginate analogs (randomly selected from a library of 211 newly synthesized analogs (represented in Figure 1)) encapsulated with a unique combination (1:2 ratio) of two different HUVECs was implanted into the IP cavity of NHPs for 4 weeks (30 capsules per material) (Figure 5C). A representative image of the capsules before implantation showed a homogenous distribution of 1.5 mm capsules (Figure 8D). Live / dead staining of the pre-implanted capsules confirms that the cells were viable before implantation into NHPs (Figure 8D). After 4 weeks, all floating capsules in the IP cavity were collected and used for material identification using NGS assay (white arrow: tissue aggregated capsule, yellow arrow: floating capsule, Figure 8E). From the 3000 implanted capsules, a total of 503 free-floating capsules were recovered and used for NGS analysis, resulting in successful identification of approximately 92.6% of the sampled capsules with high confidence (Figure 8F). The remaining 7.4% capsules could not be confidently identified due to insufficient sequencing depth (N=5, failed) or low barcode matching likelihood (N=32, low confidence). The distribution of capsules in the confidence space is plotted in Figure 8G. This study successfully demonstrated the applicability and feasibility of in vivo screening of 100 new materials in the NHP model using a dual barcoding strategy. We identified four lead hydrogels from the 100 that showed minimal fibrosis among the NHPs (Z1-A2, Z4-A11, Z1-A27, and Z2-A27, Figure 8H). All four of these hydrogels contain hydrophilic linkers. Interestingly, these four leads from the NHP screening were not identified as top 15 leads in the mouse study and therefore were not used for further application in this study.

[0207] The new lead materials screened in mouse studies could be used in a variety of coating applications to mitigate foreign body reactions and protect against immune responses. Chemically modified alginate reduces FBR in immune-competent mice in individual settings. All materials used for screening were tested under mixed conditions for high-throughput assays. Therefore, it was desired to confirm whether the top lead materials screened had immune-protective properties in individual settings. Because the small size of the capsules may induce immune responses in a short period of time, 300-400 μm sized microcapsules were tested for 2 weeks of implantation. The top 10 lead materials, one previously reported positive control hydrogel (Z1-A34), and one negative control hydrogel (SLG20) were used for this test. Two weeks after implantation into the IP cavity of mice, dark field images showed less fibrotic deposition of the lead materials than the controls (Figure 11A). The SLG20 control, in contrast to the selected lead materials including Z4-A10 and Z1-A3, induced a more elevated immune response and most of the microcapsules aggregated within the fibrotic tissue. Surface fibrosis levels were determined by imaging and RT-qPCR analysis of capsules containing macrophage and fibroblast markers (Figure 11B-11C). Immunofluorescence imaging (Figure 11B) showed that Z4-A10 and Z1-A3 had the lowest intensity of macrophage (CD68, green) and myofibroblast (α-SMA, red) markers, indicating lower levels of fibrosis, compared to the SLG20 control. Reverse transcription-PCR analysis of fibrosis markers (α-SMA and Col1a1) revealed that the lead material had significantly lower expression of both markers, compared to SLG20, indicating lower fibrosis and reduced collagen deposition on the capsule surface (Figure 11C). Among all the top leads, Z4-A10 and Z1-A3 appeared to be the most promising in preventing FBR and were therefore considered for further applications, including delivery of xenogeneic human islets in diabetic rodents (Z4-A10) and coating of medical-grade catheters (Z1-A3). These results confirmed the antifibrotic effects of the screened lead materials in individual settings and provided opportunities for various immunomodulatory applications.

[0208] Lead hydrogel restores long-term glycemia using xenogeneic human islets in an immunocompetent animal model Antifibrotic alginate (Z4-A10, Schemes 5 and 6) hydrogels of the present invention were used to encapsulate xenogeneic human islets. These formulations provide a highly porous and antifibrotic hydrogel outer membrane to allow for long-term nutrient diffusion, high islet viability, and low fibrosis in vivo. High-throughput screening in mice increased the selection pressure to identify materials that could protect densely encapsulated xenogeneic cells from rejection, using a high density cell load, approximately 30,000 HUVECs per capsule. For the diabetes correction studies described herein, a similar cell density per capsule (15K-60K cells per capsule) was maintained to evaluate the efficacy of lead formulations to allow long-term survival and protection of islets in STZ-induced C57BL / 6J mice.

[0209] Capsules of three different densities (alginate 4K IEQ / mL, alginate 8K IEQ / mL, alginate 16K IEQ / mL, Figure 12) of human islets were prepared using Z4-A10 alginate, a lead triazole-containing alginate identified in a high-throughput screen (Figures 7E-7F). Control SLG20 capsules were prepared at islet cell densities of 4K IEQ / mL and 16K IEQ / mL. Dithizone staining and live / dead imaging prior to implantation of capsule groups (Z4-A10 and control SLG20) demonstrated islet viability (Figure 13A). Z4-A10 capsules at a density of 4K IEQ / mL demonstrated long-term restoration of normoglycemia, maintaining glycemic correction up to 80 days of data recording, with fasting mean blood glucose (BG) values ​​below 250, considered BG values ​​for healthy mice (Figure 13B). However, control SLG20 alginate at the same dose (IEQ density 4K / mL) failed to maintain glycemic correction for longer than 4 weeks. Intravenous glucose tolerance test (IVGTT) performed after 4 hours fasting on day 75 showed that encapsulated islet cells restored normoglycemia to a rate comparable to that of healthy C57BL / 6J mice (Figure 13C). Furthermore, capsule images after retrieval (dark field) showed minimal fibrotic hyperplasia on the surface of Z4-A10 capsules compared to SLG20 (Figure 13D). Dithizone staining also supported long-term islet viability 80 days after transplantation (Figure 13E). Concentrations of human c-peptide, a surrogate biomarker for insulin production, were measured from serum isolated from mouse blood 80 days after transplantation. Higher c-peptide secretion levels were observed in the Z4-A10 group compared to SLG20, suggesting, without being bound by theory, improved long-term survival (Figure 13F).

[0210] In the high density encapsulation group, Z4-A10 capsules at 16K IEQ / mL concentration were able to maintain long-term glycemic control function for more than 50 days (FIG. 13G). In contrast, the control SLG20 group was unable to maintain glycemic control for 10 days or less after transplantation (FIG. 13H). These results, again without being bound by theory, suggest that Z4-A10 has an enhanced ability to protect encapsulated islets from foreign body reactions, maintaining longer graft viability and function.

[0211] Lead anti-fibrotic materials show low fibrosis when coating catheters. To test the anti-fibrotic efficacy of the newly developed small molecules in the context of other medical devices, medical grade silicone catheters were plasma treated and coated with methacryloyl modified Z1-A3 (one new hydrophilic lead) and B2-A17 (one new hydrophobic lead) (Figure 14A, Schemes 7-10). Silicone catheters are hydrophobic in nature. Therefore, one top hydrophobic lead (B2-A17) and one hydrophilic lead (Z1-A3) were selected to investigate whether hydrophilicity / hydrophobicity influences mitigation of fibrosis. XPS and ToF-SIMS were used to analyze the surface chemistry of unmodified and coated catheters, confirming successful coating of catheters (Figure 14B-14C, Figure 14A-14C).

[0212] To examine the fibrotic response in uncoated and small molecule coated catheters in a C57BL / 6J mouse model of developing fibrosis, these catheters were implanted subcutaneously in mice for 4 weeks. Histological examination and imaging of catheters excised after 4 weeks revealed that compared with the small molecule coated catheters (Met-Z1-A3 and Met-B2-A17), the unmodified catheter excisions resulted in thick dark purple tissue deposits (which are rich in collagen and indicative of fibrotic hyperplasia) between the catheter and the skin tissue (Figure 14D-14E, Figure 15D-15E). The results, without being bound by theory, indicate that the small molecule coated catheters may prevent FBR better than the unmodified catheters. It is also noteworthy that when a hydrophobic lead (Met-B2-A17) was used to coat the hydrophobic silicone catheters, it was better at preventing fibrotic deposition than one of the hydrophilic leads (Met-Z1-A3).

[0213] NMR data for the top 20 best performing alginate analogues. TIFF2024522379000039.tif41169TIFF2024522379000040.tif238170TIFF2024522379000041.tif122170

[0214] TIFF2024522379000042.tif67157Scheme 1. Schematic representation of the synthesis of B1-A51 amines. Mass Spectrometry and NMR Data: TIFF2024522379000043.tif40166

[0215] TIFF2024522379000044.tif58158Scheme 2. Schematic representation of the modification of alginate with B1-A51. NMR data: TIFF2024522379000045.tif27168 Elemental analysis: C: 34.75%, H: 8.55%, N: 3.56%.

[0216] TIFF2024522379000046.tif79141Scheme 3. Schematic representation of the synthesis of Z1-A34 amine. Mass Spectrometry and NMR Data: TIFF2024522379000047.tif49166

[0217] TIFF2024522379000048.tif75154Scheme 4. Schematic representation of the modification of alginate with Z1-A34. NMR data: TIFF2024522379000049.tif19170 Elemental analysis: C: 35.67%, H: 4.34%, N: 5.08%, O: 33.50%.

[0218] TIFF2024522379000050.tif74154Scheme 5. Schematic representation of the synthesis of Z4-A10 amine. Mass Spectrometry and NMR Data: TIFF2024522379000051.tif56170

[0219] TIFF2024522379000052.tif80157Scheme 6. Schematic representation of the modification of alginate with Z4-A10. NMR: TIFF2024522379000053.tif12158 Elemental analysis: C: 40.45%, H: 5.60%, N: 6.22%.

[0220] TIFF2024522379000054.tif67161Scheme 7. Schematic representation of the synthesis of Z1-A3 amines. Mass Spectrometry and NMR Data: TIFF2024522379000055.tif41168

[0221] TIFF2024522379000056.tif78170Scheme 8. Schematic representation of the synthesis of Met-Z1-A3. Mass Spectrometry and NMR Data: TIFF2024522379000057.tif62169

[0222] TIFF2024522379000058.tif64165Scheme 9. Schematic representation of the synthesis of B2-A17 amine. Mass Spectrometry and NMR Data: TIFF2024522379000059.tif48170

[0223] TIFF2024522379000060.tif73166Scheme 10: Schematic representation of the synthesis of Met-B2-A27. Mass Spectrometry and NMR Data: TIFF2024522379000061.tif63163

[0224] TIFF2024522379000062.tif63170 Scheme 11: Schematic representation of the synthesis of Z2-A19.

[0225] All of the compounds, formulations, and methods disclosed and claimed herein can be made and carried out without undue experimentation in light of this disclosure.Although the compounds, formulations, and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to one skilled in the art that variations may be applied to the compounds, formulations, and methods, and to the steps or order of steps of the methods described herein, without departing from the concept, spirit, and scope of the present invention.More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein with the same or similar results.All such similar substitutes and modifications that are apparent to one skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0226] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024522379000063.tif63147

Claims

1. The following: A compound represented by: wherein: m and n are the number of repeating units having a molecular weight of from about 50,000 Daltons to about 500,000 Daltons.

2. A compound of the following formula: A-L-R 1 (I) or a pharmaceutically acceptable salt thereof, wherein: wherein: A is a polymer; L is a linker of the following formula: NR a X 1 (CH 2 CH 2 O) o wherein: and R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; o is 2, 3, 4, or 5; or a linker of the following formula: X 1 is an alkandiyl (C≦8) or a substituted alkandiyl (C≦8) or; wherein: NR b (CH 2 ) p X 2 p is 1, 2, or 3; and R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; a group of: wherein: X 2 is an arene diyl (C≦12) or a substituted arene diyl (C≦12) ; R 1 is alkynyl (C≤6) - substituted aryl (C≤12); haloaryl (C≦12) ; S - containing heteroaryl (C≦12) ; cycloalkyl (C≤12); 2 - pyridinyl; substituted S - containing heteroaryl (C≦12) ; alkyl (C≦6) ; haloalkyl (C≦6) ; or alkenyl (C≦6) substituted aryl (C≦12) ; aralkyl (C≦12) ; substituted aralkyl (C≦12) ; heterocycloalkyl (C≦12) ; substituted heterocycloalkyl (C≦12) ; 3 - aminophenyl; 4 - alkoxy (C≦6) substituted aryl (C≦12) ; or the following formula: X 3 OR 2 a compound or a pharmaceutically acceptable salt thereof.

3. X 3 is an alkanediyl (C≦8) or a substituted alkanediyl (C≦8) and; R 2 is aryl (C≦12) or substituted aryl (C≦12) and is The following: further defined as, wherein: A-L-R 1 (I) A is a polymer; L is a linker of the following formula: wherein: m is 2, 3, 4, or 5; and NR a X 1 (CH 2 CH 2 O) m a group of: wherein: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; the compound according to Claim 2, or a pharmaceutically acceptable salt thereof. X 1 is an alkanediyl (C≦8) or a substituted alkanediyl (C≦8) and; R 1 is cycloalkyl (C≦12) ; haloaryl (C≦12) ; S-containing heteroaryl (C≦12) ; substituted S-containing heteroaryl (C≦12) ; alkyl (C≦6) ; haloalkyl (C≦6) ; alkenyl (C≦6) ; or alkyne (C≦6) substituted aryl (C≦12) ; 3-aminophenyl; 4-alkoxy (C≦6) substituted aryl (C≦12) ; or the following formula: X 3 OR 2

4. The following: X 3 is an alkanediyl (C≦8) or a substituted alkanediyl (C≦8) and; R 2 is aryl (C≦12) or substituted aryl (C≦12) and is further defined as, wherein: A is a polymer; A-L-R 1 (I) L is a linker of the following formula: wherein: and or a linker of the following formula: NR a X 1 (CH 2 CH 2 O) m wherein: n is 1, 2, or 3; R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; m is 2, 3, 4, or 5; and X 1 is an alkanediyl (C≦8) or a substituted alkanediyl (C≦8) or; the compound according to Claim 2, or a pharmaceutically acceptable salt thereof. NR b (CH 2 ) n X 2

5. The compound according to Claim 2, wherein the polymer comprises one or more sugar repeating units. R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ;

6. (i) The repeating unit has the following formula: wherein: X 2 is an arenediyl (C≦12) or a substituted arenediyl (C≦12) ; R 1 is a haloaryl (C≦12) ; aralkyl (C≦12) ; substituted aralkyl (C≦12) ; heterocycloalkyl (C≦12) ; substituted heterocycloalkyl (C≦12) ; 2-pyridinyl; 3-aminophenyl, m is the number of repeating units having a molecular weight of from about 50,000 Daltons to about 500,000 Daltons, or (ii) the polymer comprises repeating units of the following formula: wherein: R 3, R 3 ', R 4, or R 4'are each independently hydrogen or hydroxy; R 5 is hydroxy, alkoxy (C≦8), substituted alkoxy (C≦8), or a covalent bond to the linker; R 5'is a covalent bond to the linker; and R 3 or R 4 is independently hydrogen or hydroxy; R 5 is hydroxy, alkoxy (C≦8) , substituted alkoxy (C≦8) , or a covalent bond to the linker; and m and n are the number of repeating units having a molecular weight of from about 50,000 Daltons to about 500,000 Daltons, the compound according to Claim 5.

7. The compound according to Claim 2, wherein the polymer is an acrylate polymer.

8. The compound according to Claim 7, wherein the polymer is a methacrylate polymer.

9. The compound according to Claim 2, wherein o is 2, 3, or 4.

10.

11.

12.

13. (a) R 1 is cycloalkyl (C≦12); (c) R 1 is 3-aminophenyl; ​ ​ ​ R a The compound according to claim 2, wherein R is hydrogen. ​ X 1 is an alkanediyl (C≦6) The compound according to claim 2, wherein ​ X 1 is -CH 2 CH 2 - and is the compound according to claim 11. ​ (b) R 1 is a haloaryl (C≦12) ; ​ (d) R1 is S-containing heteroaryl (C≤12) or substituted S-containing heteroaryl (C≤12); (e) R1 is alkyl (C≤6), haloalkyl (C≤6), alkenyl (C≤6), or alkyne (C≤6) substituted aryl (C≤12); (f) R1 is a group of the following formula: X3OR2 wherein: X3 is alkanediyl (C≤8) or substituted alkanediyl (C≤8); R2 is aryl (C≤12) or substituted aryl (C≤12); (g) R1 is 4-alkoxy (C≤6) substituted aryl (C≤12); (h) R1 is heterocycloalkyl (C≤12) or substituted heterocycloalkyl (C≤12); or, (i) R1 is aralkyl (C≤12) or substituted aralkyl (C≤12), the compound according to claim 2.

14.

15. R 1 The compound according to claim 13, wherein R is cyclopropyl.

16. R 1 The compound according to claim 13, wherein R is chlorophenyl, bromophenyl, or fluorophenyl.

17. R 1 The compound according to claim 13, wherein R is 2-bromophenyl, 4-chlorophenyl, 2-fluorophenyl, or 4-fluorophenyl.

18. R 1 is a heteroaryl containing S (C≦12) The compound according to claim 13, wherein

19. R 1 The compound according to claim 13, wherein R is 2-thienyl or 3-thienyl.

20. R 1 is an alkyne (C≦6) substituted aryl (C≦12) The compound according to claim 13, wherein

21. R 1 The compound according to claim 13, wherein R is 3-ethynyl-phenyl.

22. R 1 is alkyl (C≦6) substituted aryl (C≦12) The compound according to claim 13, wherein it is

23. R 1 The compound according to claim 13, wherein R is 3-methylphenyl or 4-methylphenyl.

24. R 1 is haloalkyl (C≦6) substituted aryl (C≦12) The compound according to claim 13, wherein

25. R 1 The compound according to claim 13, wherein R is 4-trifluoromethylphenyl.

26. X 3 is an alkanediyl (C≦8) The compound according to claim 13, wherein X is an alkanediyl

27. X 3 is -CH 2 - and is the compound according to claim 13.

28. R 2 is a substituted aryl (C≦12) The compound according to claim 13, wherein

29. R 2 The compound according to claim 13, wherein R is 4-aminophenyl.

30. R 1 The compound according to claim 13, wherein R is 4-ethoxyphenyl.

31. R 1 is a heterocycloalkyl (C≦12) The compound according to claim 13, wherein:

32. R 1 The compound according to claim 13, wherein R is thiomorpholine dioxide.

33. R 1 is an aralkyl (C≦12) The compound according to claim 13, wherein

34. o is 2, Ra is hydrogen, X1 is alkanediyl (C≤6), and R1 is cycloalkyl (C≤12), the compound according to claim 2. R 1 The compound according to claim 13, wherein R is 2-phenylethyl.

35. Rb is hydrogen, and / or p is 1 or 2, the compound according to claim 2.

36.

37. A method for detecting fibrosis in a sample, the method comprising the steps of exposing the sample to one or more compounds according to any one of claims 1 to 38 and measuring reactivity.

38.

37. X 2 is an arenediyl (C≦12) The compound according to claim 2

39. A medical device coated with a compound according to any one of claims 1 to 38. X 2 The compound according to claim 37, wherein X is benzenediyl.

40.

41.

42.

43.

44. The medical device according to claim 40, which is a transplantable device, a cardiac pacemaker, a catheter, a needle injection catheter, a thrombus filter, a vascular graft, a balloon, a stent graft, a biliary stent, an intestinal stent, a bronchial stent, an esophageal stent, a ureteral stent, an aneurysm filling coil or other coil device, a surgical repair mesh, a breast implant, a silicone implant, PDMS, a transmyocardial revascularization device, a percutaneous myocardial revascularization device, a prosthesis, an organ, a blood vessel, an aorta, a heart valve, a tube, an organ replacement part, an implant, a fiber, a hollow fiber, a membrane, a textile, a blood reservoir, a blood container, a titer plate, an adsorption medium, a dialyzer, a connecting component, a sensor, a valve, an endoscope, a filter, a pump chamber, or another medical device intended to have blood compatibility, or another medical device used in cytokine therapy including cancer, diabetes, ischemia, antibacterial, hemophilia, stroke, blood disorders, or human modified cells.

42. (a) A capsule, a transplantable polymer block, a 3D printed block, a 3D printed gel, or a polymer encapsulated device, or (b) A catheter, the medical device according to claim 41.

43. (i) The polymer encapsulated device further includes a shape selected from spherical, square, noodle-shaped, needle-shaped, rectangular, and cylindrical, or (ii) The transplantable capsule is a microcapsule, the medical device according to claim 42.

44. The medical device according to claim 40, which has less fibrosis than a medical device without a coating.

45. The medical device according to claim 44, which is immunoprotective compared to a medical device without a coating.

46. The medical device according to claim 45, wherein the immunoprotection results in a lower foreign body reaction.

47. A pharmaceutical composition comprising (A) a compound according to any one of claims 1 to 38; and (B) an excipient .

48. The pharmaceutical composition according to claim 47, wherein the compound is crosslinked.

49. The pharmaceutical composition according to claim 48, wherein the crosslinked compound is crosslinked by a covalent bond.

50. The pharmaceutical composition according to claim 47, further comprising a biological material.

51. The pharmaceutical composition according to claim 50, wherein the biological material is encapsulated in the compound.

52. The pharmaceutical composition according to claim 51, wherein the biological material is a cell.

53. (a) The cell is a cell from a heterologous tissue, a cell from a cadaver, a stem cell, a cell derived from a stem cell, a cell from a cell line, a primary cell, a reprogrammed cell, a reprogrammed stem cell, a cell derived from a reprogrammed stem cell, a genetically engineered cell, or a combination thereof; (b) The cell is a human cell; or, (c) The cell is an insulin-producing cell, the pharmaceutical composition according to claim 52.

54. The pharmaceutical composition according to claim 53, wherein the cell is an islet cell.

55. The medical device according to claim 40, for use in a method of treating or preventing a disease or disorder.

56. (a) Resulting in a lower foreign body reaction, and / or (b) Resulting in less fibrosis, the medical device according to claim 55.

57. The pharmaceutical composition according to claim 47, for use in a method of treating or preventing a disease or disorder.

58. (a) Resulting in a lower foreign body reaction, and / or (b) Resulting in less fibrosis, the pharmaceutical composition according to claim 57.

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