Nanogels conjugated with cell-penetrating peptide as drug delivery vehicle for treating urinary tract infections

Abstract

Among hospital–acquired infections, Pseudomonas aeruginosa-associated urinary tract infections (UTIs) are mainly caused by indwelling urethral catheters (catheter-associated UTIs or CAUTIs) and are difficult to treat, resulting in high rates of morbidity among hospitalized patients. While antibiotics can successfully treat bacteria in the bladder lumen, they are inefficient at crossing stratified urothelium plasma membranes to kill persistent intracellular bacterial communities (IBCs). Herein, UTI IBCs are targeted by locally delivering the antibiotic gentamicin and / or the steroid hormone estradiol via polymeric nanogels conjugated with a cell-penetrating peptide Cys-Gly-Lys-Arg-Lys (CGKRK - SEQ ID NO:1). This approach delivered more intracellular gentamicin compared to drug delivered in solution in vitro and more estradiol compared to conventional methods. In an acute UTI murine model, the nanogel cell-penetrating peptide drug delivery system facilitated the transport of gentamicin into the urothelium and resulted in > 90% clearance of a uropathogenic P. aeruginosa clinical strain in vivo.

Description

Attorney ref 340541: 12-25 WONANOGELS CONJUGATED WITH CELL-PENETRATING PEPTIDE AS DRUG DELIVERY VEHICLE FOR TREATING URINARY TRACT INFECTIONSSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grant numbers 1R21A1154360-01 and T32AI052066 awarded by the National Institute of Allergy and Infectious Diseases, grant number 1R21DE032135-01 A1 awarded by the National Institutes of Health - National Institute of Dental and Craniofacial Research (NIH-NIDCR), and grant numbers BX0002073 and IK6BX005384 awarded by the Veterans Administration. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from United States Provisional Patent Application No. 63 / 762,508 filed February 24, 2025, which is incorporated by reference herein to the extent that there is no inconsistency with the present disclosure.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (“340541_12-25_WO _seq_listing.xml”; Size: 1,821 bytes; and Date of Creation: February 23, 2026) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] Urinary tract infections (UTIs) are some of the most common and impactful infections seen in both inpatient and outpatient settings. UTIs disproportionally affect children, women, and the elderly and are responsible for a significant worldwide economic burden [1 , 2], In addition to medical costs, patients who suffer from UTIs typically experience a compromised quality of life [3], Left untreated, UTIs can cause kidney damage, sepsis, and even death [4],

[0005] Uncomplicated UTIs are commonly caused by uropathogenic Escherichia coli. However, rising rates of Pseudomonas aeruginosa (P. aeruginosa) infections have been reported in recurrent UTIs (rUTIs), hospital-acquired UTIs, and catheter-associated UTIs (CAUTIs) [5, 6], P. aeruginosa can invade the urothelium and survive intracellularly via the formation of persistent intracellular bacterial communities (IBCs) [7, 8], While antibiotic regimens can successfully treat bacteria in the bladder lumen, they are typically inefficient inAttorney ref 340541: 12-25 WOcrossing the stratified urothelium plasma membranes, making P. aeruginosa IBCs difficult to treat. Repeated use of antibiotic therapies has also contributed to the growing bacterial resistance, resulting in higher morbidities [4, 8], There is an urgent need to develop alternative treatment strategies to treat and combat UTIs and rUTIs. Drug delivery modalities that can effectively deliver therapeutics by crossing urothelial plasma membranes to access IBCs may provide an effective mechanism of eliminating UTIs while curbing the excessive use of antibiotics to fight infections.

[0006] Previous studies have assessed the efficacy of nanoparticles (e.g., carbon-, metallic-, and lipid-based materials) for drug delivery for UTI treatment [9-11], Although they have reduced bacterial infection, the relatively large size (100 nm - 500 nm), morphology, and rigidity of these particles prevents them from crossing membranes and stratified cell layers. In contrast, polymeric nanogels can be synthesized as soft, spherical, crosslinked networks that can reversibly swell and collapse [12-14], Furthermore, nanogels are more biocompatible than nanoparticles, have a flexible size under 100 nm, and are able to encapsulate, stabilize, and deliver a wide range of therapeutics in a targeted manner

[0012] , The inventors have extensively studied the parameters that control the size, morphology, and surface properties by monomer selection and choice of solvent / solvent volume that can facilitate small-molecule delivery in highly tailored, biocompatible nanogels

[0015] , Additionally, previous work has shown that intravesically (l-VESIC)-delivered nanogels conjugated with the cell-penetrating peptide (CPP), Cys-Gly-Lys-Arg-Lys (CGKRK- SEQ ID NO:1), successfully penetrate the urothelial cells at multiple levels and deliver encapsulated contents in a healthy murine model

[0016] , (See also U.S. Pat. No. 11,642,319). Yet, no extensive work has reported the use of targeted nanogels with a CPP for antibiotic delivery in a UTI treatment model.

[0007] Hence, a biocompatible nanogel was created conjugated with CPP that could be used as l-VESIC drug delivery vehicles to meet the challenge of delivering an antibiotic directly into urothelial cells in order to target IBCs in a UTI murine model. In the examples below, the hydrophilic antibiotic gentamicin (GEN) was selected, owing to its long-considered impermeability to urothelial cell membranes

[0017] , The examples described herein were aimed to determine the efficacy and feasibility of specially designed nanogels conjugated with CPP to encapsulate GEN, cross the urothelial cell plasma membranes, and deliver therapeutic doses to eradicate P. aeruginosa IBCs in vitro and in an acute UTI murine model.SUMMARY OF THE INVENTIONAttorney ref 340541: 12-25 WO

[0008] The present invention provides compositions and methods for treating microbial infections, particularly bacterial infections caused by persistent intracellular bacterial communities (IBCs). In particular, one aspect of the invention provides compositions and methods for treating urinary tract infections (UTIs).

[0009] In an embodiment, the present invention provides a therapeutic composition comprising: a) a nanogel comprising a plurality of hydrogel particles having an average diameter from 1 nm to 1,000 nm, where each of the hydrogel particles comprise one or more crosslinked polymers; b) one or more cell-penetrating peptides (CPPs) conjugated to the hydrogel particles of the nanogel; and c) one or more therapeutic agents, wherein the one or more therapeutic agents are at least partially encapsulated by the nanogel. Preferably, the one or more therapeutic agents comprise one or more antimicrobial agents, one or more steroid hormones, or combinations thereof.

[0010] As used herein, nanogels refer to three-dimensional, polymer-based, crosslinked hydrogel materials in the nanoscale size range formed by crosslinked polymer networks that are able to swell in water without dissolving (see, for example, Ferozekhan et al., Cureus, 2024, 16(9): e68633). Preferably, the nanogels are biocompatible and are able to administered to a human patient resulting in little to no cytotoxicity or adverse effects.

[0011] Nanogels may comprise a variety of naturally occurring polymers, synthetic polymers, and combinations thereof, including but not limited to polymerized monovinyl and divinyl monomers. In an embodiment, the plurality of hydrogel particles comprise a plurality of crosslinked monomers of chitosan, alginate, hyaluronic acid, heparin, chondroitin, collagen, pullulan, acrylic acid, polyacrylic acid, poly(methacrylic acid), 2-hydroxyterephthalic acid, 2- hydroxyethyl acrylate (HEA), tetra(ethylene) glycol dimethacrylate (TTEGDMA), tetraethyl dimethacrylate, polyethylenimine, polyethylene glycol, polyvinylpyrrolidone, poly(lactic-co-glycolic) acid, polyethylene glycol) methacrylate, polyethylene gylcol) dimethacrylate, stearyl acrylate (SA), or combinations thereof.

[0012] The plurality of hydrogel particles may comprise one or more types of monomers, two or more types of monomers, three or more types of monomers, four or more types of monomers, or five or more types of monomers. The molar ratio of each type of monomers in the nanogel other are variable and may adjusted in order to effectively encapsulate the desired therapeutic agent.

[0013] In an embodiment, the plurality of hydrogel particles comprises crosslinked monomers of 2-hydroxyterephthalic acid, tetraethyl dimethacrylate, and acrylic acid (i.e., anAttorney ref 340541: 12-25 WOHTA nanogel), preferably, in a molar ratio of 60(± 5): 20(± 5): 20(± 5). In an alternative embodiment, the plurality of hydrogel particles comprises crosslinked monomers of stearyl acrylate (SA), polyethylene glycol) methacrylate, and polyethylene gylcol) dimethacrylate, preferably in a molar ratio of 25(± 5): 55(± 5): 20(± 5). In an alternative embodiment, the plurality of hydrogel particles comprises crosslinked monomers of acrylic acid, 2-hydroxyethyl acrylate (HEA), and tetra(ethylene) glycol dimethacrylate (TTEGDMA), preferably in a molar ratio of 60(± 5): 20(± 5): 20(± 5).

[0014] Characteristics such as size, charge, porosity, amphiphilicity, softness, and degradability can be fine-tuned by varying the chemical composition of the nanogels as well as the degree of crosslinking as is known in the art. The nanogels may be hydrophilic, hydrophobic, or amphiphilic depending on the therapeutic agent to be encapsulated.Preferably, the nanogels are hydrophilic or amphiphilic. Optionally, the nanogel is able to absorb at least 25% w / w of water, at least 50% w / w of water, at least 60% w / w of water, at least 80% w / w of water, at least 90% w / w of water.

[0015] In an embodiment, nanogels comprise spherical and / or spheroid hydrogel particles but may optionally comprise different shapes and combinations thereof. In an embodiment, the nanogel comprises spherical and / or spheroid hydrogel particles having an average diameter of about 1 nm - 1 ,000 nm, about 1 nm - 800 nm, about 1 nm - 100 nm, about 1 nm - 50 nm, about 5 nm - 600 nm, about 10 nm - 500 nm, about 10 nm - 250 nm, about 10 nm - 100 nm, about 20 nm - 200 nm, about 50 nm - 150 nm, or any range formed from any two of the foregoing values.

[0016] In an embodiment, the nanogels comprise spherical and / or spheroid hydrogel particles having a crosslinked polymer core and are conjugated to a plurality of peptides extending from the core. Preferably, the plurality of peptides comprise one or more cellpenetrating peptide (CPP). Cell-penetrating peptides (CPPs) are short amino acid sequences, typically between 5 and 30 amino acids, able to facilitate transport of molecules across cellular membranes, including but not limited to direct translocation and / or endocytosis. In an embodiment, the CPP is cationic, amphipathic, or hydrophobic, and optionally comprises arginine and lysine residues.

[0017] CPPs suitable with the present invention include, but are not limited to, CPPs currently known in the art (see, for example, Bottens et al., Cancers, 2022, 14(22): 5546). In an embodiment, the one or more CPPs comprise the trans-activator of transcription (TAT) protein identified from the HIV1 virus, transportan, pVEC, penetratin, Cys-Gly-Lys-Arg-LysAttorney ref 340541: 12-25 WO(CGKRK - SEQ ID NO: 1), and combinations thereof. Preferably, the CPP is able to covalently link with residual acrylate groups on the hydrogel particles of the nanogel, including but not limited to linking with residual acrylate groups through a cysteine residue in the CPP. In an embodiment, the one or more CPPs comprise at least 5 amino acids and comprises equal to or less than 30 amino acids, equal to or less than 20 amino acids, equal to or less than 10 amino acids, or equal to or less than 8 amino acids, or any intermediate range formed from the foregoing values. In an embodiment, the one or more CPPs comprise 5 amino acids. Preferably, the one or more CPPs comprise a peptide having the sequence CGKRK (SEQ ID NO: 1). In an embodiment, the one or more CPPs comprise a peptide consisting of the sequence CGKRK (SEQ ID NO: 1).

[0018] The nanogel conjugated to the one or more CPPs is able to encapsulate and transport one or more therapeutic agents across cellular membranes. Such therapeutic agents include, but are not limited to, nucleic acids (including DNA, siRNA), proteins (including therapeutic proteins), and small bioactive molecules. In an embodiment, the one or more therapeutic agents comprise one or more antimicrobial agents, including an antibacterial agent, an anti-viral agent, an anti-fungal agent, an anti-parasitic agent, or combinations thereof. Optionally, the one or more antimicrobial agents are hydrophilic. In an embodiment, the one or more therapeutic agents comprise one or more steroid hormones, including but not limited to estrogens such as estradiol, estriol, estrone, and pharmaceutical salts and formulations thereof.

[0019] In an embodiment, the one or more antimicrobial agents comprise an anti-bacterial agent able to reduce or eliminate intracellular bacterial communities (IBCs), such as those of Pseudomonas aeruginosa and / or Escherichia coli. Optionally, the one or more selected molecules comprise an aminoglycoside antibiotic agent able to treat infections caused by Gram-negative bacteria. In an embodiment, the one or more antimicrobial agents comprises gentamicin (GEN), tobramycin, amikacin, neomycin, plazomicin, paromomycin, streptomycin, nitrofurantoin, trimethoprim / sulfamethoxazole (Bactrim™), floxacins (fluoroquinolones such as ciprofloxacin), cephalosporins, penicillins, or combinations thereof, including any pharmaceutical salts and formulations thereof. Preferably the antibiotic is gentamicin (GEN).

[0020] In an embodiment, the one or more steroid hormones comprise estradiol, estriol, estrone, or combinations thereof, including any pharmaceutical salts and formulations thereof. Preferably, the one or more steroid hormones comprises estradiol orAttorney ref 340541: 12-25 WOpharmaceutical salts and formulations thereof. Except at its molecular termini, estradiol is both nonpolar and hydrophobic and insoluble in water.

[0021] In an embodiment, the one or more therapeutic agents comprise a combination of one or more antimicrobial agents and one or more steroid hormones, preferably gentamicin and estradiol, including any pharmaceutical salts and formulations thereof.

[0022] In an embodiment, the present invention provides a method fora urinary tract infection (UTI) in a patient comprising administering an effective amount of a therapeutic composition to a patient in need thereof, where the therapeutic composition comprises: a) a nanogel comprising a plurality of hydrogel particles having an average diameter from 1 nm to 1,000 nm, wherein each of the hydrogel particles comprise one or more crosslinked polymers; b) one or more cell-penetrating peptides (CPPs) conjugated to the hydrogel particles of the nanogel; and c) one or more therapeutic agents, where the one or more therapeutic agents comprises one or more antimicrobial agents, one or more steroid hormones, or combinations thereof, and where the one or more therapeutic agents are at least partially encapsulated by the nanogel. Administering the effective therapeutic amount of the antimicrobial composition reduces or eliminates the bacteria causing the UTI.

[0023] The therapeutic compositions administered to the patient and components thereof are the same as described above. Preferably, the therapeutic composition administered to the patient comprises a combination of one or more antimicrobial agents and one or more steroid hormones, preferably gentamicin and estradiol, including any pharmaceutical salts and formulations thereof.

[0024] In an embodiment, the therapeutic composition comprises between about 0.001 mg / mL and 1 ,000 mg / ml of the one or more therapeutic agents (including but not limited to gentamicin and / or estradiol), between about 0.01 mg / mL and 100 mg / ml of the one or more therapeutic agents, between about 0.1 mg / mL and 60 mg / ml of the one or more therapeutic agents, between about 1 mg / mL and 50 mg / ml of the one or more therapeutic agents, between about 2 mg / mL and 40 mg / ml of the one or more therapeutic agents, between about 4 mg / mL and 20 mg / ml of the one or more therapeutic agents, between about 5 mg / mL and 10 mg / ml of the one or more therapeutic agents, or any range formed from any two of the foregoing values.

[0025] In an embodiment between about 1 pg and 10 g of the one or more therapeutic agents (including but not limited to estradiol) are administered to the patient per day,Attorney ref 340541: 12-25 WObetween about 5 g and 1 g of the one or more therapeutic agents, between about 10 pg and 500 mg of the one or more therapeutic agents, between about 50 pg and 250 mg of the one or more therapeutic agents, between about 100 pg and 100 mg of the one or more therapeutic agents, between about 100 pg and 10 mg of the one or more therapeutic agents, between about 250 pg and 5 mg of the one or more therapeutic agents, between about 400 pg and 4 mg of the one or more therapeutic agents, between about 500 pg and 2 mg of the one or more therapeutic agents, between about 1 mg and 1.5 mg of the one or more therapeutic agents, or any range formed from any two of the foregoing values.

[0026] In an embodiment, between about 1 pg / kg and 1 g / kg of the one or more therapeutic agents (including but not limited to one or more of the antibiotics, such as gentamicin) are administered to the patient per day, between about 10 pg / kg and 500 mg / kg of the one or more therapeutic agents, between about 100 pg / kg and 200 mg / kg of the one or more therapeutic agents, between about 500 pg / kg and 100 mg / kg of the one or more therapeutic agents, between about 1 mg / kg and 50 mg / kg of the one or more therapeutic agents, between about 2 mg / kg and 20 mg / kg of the one or more therapeutic agents, between about 3 mg / kg and 10 mg / kg of the one or more therapeutic agents, between about 4 mg / kg and 7 mg / kg of the one or more therapeutic agents, or any range formed from any two of the foregoing values.

[0027] The therapeutic composition may be administered using any means as is known in the art, including but not limited to oral administration, transdermal patches, gels, sprays, vaginal creams, intramuscular injections, subcutaneous injections, intravenous administration, and bladder instillation. Optionally, the therapeutic composition is administered directly to the bladder of the patient, such as through a catheter and bladder instillation. Administering the therapeutic compositions of the present invention directly to the bladder is beneficial for treating UTIs in that the surface of the bladder does not have to be scraped or the cells damaged in order to deliver the therapeutic agents. Instead, the nanogel conjugated with the CCP is able to facilitate absorption of the therapeutic agents by the urothelial cells of the patient.

[0028] In an embodiment, administration of the effective therapeutic amount of the antimicrobial composition reduces an amount of intracellular bacterial communities (IBCs) in the patient by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 95%, or by at least 99%. In an embodiment, the IBCs in the patient comprise Pseudomonas aeruginosa or Escherichia coli, preferably Pseudomonas aeruginosa.Attorney ref 340541: 12-25 WO

[0029] In some instances, the UTI is a recurring UTI that was resistant or unaffected by a initial conventional antibacterial treatment. Thus, an embodiment comprises administering an initial treatment to the patient and administering the effective therapeutic amount of the antimicrobial composition to the patient at least 48 hours (at least 96 hours, at least 1 week, at least 2 weeks, or at least 1 month) after the initial treatment was ineffective in reducing or eliminating the bacteria causing the UTI.BRIEF DESCRIPTION OF THE FIGURES

[0030] Figure 1. Overview for the composition and synthesis of HTA-CPP in an embodiment of the invention. Composition of HTA nanogel synthesized after free-radical solution polymerization followed by conjugating the cell-penetrating peptide (CPP) Cys-Gly-Lys-Arg-Lys (CGKRK - SEQ ID NO: 1) onto the nanogel’s surface nanogel (HTA-CPP) for loading and delivering the gentamicin antibiotic in the HTA-CPP.

[0031] Figure 2. Gentamicin release kinetics from the HTA and HTA-CPP. Gentamicin released from the HTA-CPP (squares) and HTA (circles) at 37°C in PBS via dialysis method. Gentamicin release was measured by spectroscopy of fluorescently derivatized GEN as described in the Materials and Methods every 2 h to 12 h, 24 h and 96 h. Error bars are the standard deviation for N = 3.

[0032] Figure 3. Effects of HTA-CPP concentrations on urothelial cell viability. The metabolic activity of human urothelial cells (HTB-9) was determined by MTT assay after 24 h exposure to the HTA conjugated to the CPP (HTA-CPP). Cells were exposed to 1 % (v / v) Triton X-100 as a positive control. Cells that were not exposed to HTA-CPP (0 pg) was the negative control. Percent metabolic activity was determined as described in Material and Methods. Error bars are the standard devitation for N = 3. One-way ANOVA woth post-hoc Dunnett’s multiple comparison test; **“, p < 0.0001.

[0033] Figure 4. Gentamicin absorption by human urothelial cells delivered as free drug vs. encapsulated in HTA-CPP. The amount of intracellular radioactive3H-gentamicin in human urothelial cells (HTB-9) by counts-per-minute from lysed cells after3H-gentamicin was delivered either as a free drug in solution (GEN, left bar) or by HTA-CPP (HTA-CPP+GEN, right bar) after 4 h. Student t-test; ", p < 0.01.

[0034] Figure 5. Effects of HTA-CPP on murine bladders. Representative images of hematoxylin and eosin-stained murine bladders that were (A) untreated and treated withAttorney ref 340541: 12-25 WOHTA-CPP (250 pg), after (B) 24 h, and (C) 48 h (magnification 100X). Representative images of harvested infected murine bladders of (D) untreated (control) and post (E) 15min and (F) 24 h intravesical treatment with rhodamine B payload from HTA-CPP. Rhodamine channel for rhodamine B molecule (bottom; lex= 558 nm; lem= 575 nm) and Hoechst channel for nuclei (top; lex= 353 nm; lem= 485 nm).

[0035] Figure 6. Intracellular P. aeruginosa within murine bladder. Representative images of (A) P. aeruginosa UR34-GFP infected murine bladders at 10 dpi and (C) Z-stack images of P. aeruginosa intracellular bacterial community. FITC channel for UR34-GFP and DAPI channel for nuclei of murine bladder.

[0036] Figure 7. Effect of subcutaneous gentamicin, HTA+GEN and HTA-CPP+GEN on murine P. aeruginosa infected bladders. Bacterial quantification from uropathogenic P. aeruginosa (UR34-GFP)-infected murine bladders at 3 dpi for (A) untreated (N = 9) and treated subcutaneous (Sub-Q) delivery 1000 pg GEN (N = 9), treated HTA-CPP+GEN (N = 16) by l-VESIC (1000 pg GEN delivered), and for (B) treated HTA+GEN (N = 13) intravesically (l-VESIC) (1000 pg GEN delivered) in comparison to HTA-CPP+GEN group. Box and whisker plot with horizontal line for the median between the interquartile range and minimum / maximum extremes. Kruskal-Wallis one-way ANOVAwith post-hoc Dunn’s multiple comparison test against untreated and Mann Whitney test respectively; **, p < 0.01; **** p < 0.0001.

[0037] Figure 8. Immunohistochemistry (IHC) staining for macrophages on murine bladders treated with HTA-CPP. Representative images of murine bladder tissue that were untreated and treated with HTA-CPP (250 pg) after 24 h and 48h (magnification 200X) and stained for macrophage marker F4-80. Spleen was stained for positive and negative control for F4-80 stain. Black bar is 100 microns scale.

[0038] Figure 9. Immunohistochemistry (IHC) staining for neutrophils on murine bladders treated with HTA-CPP. Representative images of murine bladder tissue that were untreated and treated with HTA-CPP (250 pg) after 24 h and 48h (magnification 200X) and stained for neutrophils marker myeloperoxidase (MPO). Spleen was stained for positive and negative control for F4-80 stain. Black bar is 100 microns scale.

[0039] Figure 10. Effect of subcutaneous gentamicin, HTA+GEN, and HTA-CPP+GEN on murine P. aeruginosa bacteriuria and kidneys. Quantification of uropathogenic P.Attorney ref 340541: 12-25 WOaeruginosa (UR34-GFP) in urine (bacteriuria) at (A) 1 dpi and (B) 2 dpi and (C) in kidneys at 3 dpi of infected mice groups untreated (N = 10) and treated with 1000 pg gentamicin (GEN) via subcutaneous (Sub-Q; N = 4) or via intravesical (l-VESIC) delivery by HTA (N = 2) or HTA-CPP (N =10). Box and whisker plot with horizontal line for the median between the interquartile range and minimum / maximum extremes. Kruskal-Wallis one-way ANOVA.

[0040] Figure 11. Molecular weights and structures of monomer and initiators used to generate nanogel compositions containing estradiol in an embodiment of the invention.

[0041] Figure 12. Graphs showing estradiol release overtime in phosphate buffered saline over a period of 12 days. Error bars are standard deviations of triplicates.

[0042] Figure 13. Graph of an MTT assay showing that cell viability upon exposure to different amounts of the SA: HEMA10:PEGDMA750 nanogel (SHP NG) are comparable to a control up to 1000pg.

[0043] Figure 14. Graph illustrating in vivo mouse model where 125 pg of estradiol was injected intravascularly every 3 days for 2 weeks until the harvest date. Following the harvest, the bladders were sectioned in half, homogenized, serially diluted, and plated on LB agar to quantify CFUs.DETAILED DESCRIPTION OF THE INVENTION

[0044] Hospital-acquired infections, Pseudomonas aeruginosa-associated urinary tract infections (UTIs) are mainly caused by indwelling urethral catheters (catheter-associated UTIs or CAUTIs) and are difficult to treat, resulting in high rates of morbidity among hospitalized patients. While antibiotics can successfully treat bacteria in the bladder lumen, they are inefficient in crossing stratified urothelium plasmal membranes to kill persistent intracellular bacterial communities (IBCs).

[0045] In an embodiment, the present invention provides an approach to target UTI IBCs by locally delivering the antibiotic gentamicin via polymeric nanogels conjugated with a cellpenetrating peptide Cys-Gly-Lys-Arg-Lys (CGKRK - SEQ ID NO:1), and targeted delivery of gentamicin via the nanogels conjugated with cell-penetrating peptide. This novel approach delivered ~36% more intracellular gentamicin compared to drug delivered in solution in vitro. In an acute UTI murine model, the nanogel cell penetrating peptide drug delivery system facilitated the transport of gentamicin into the urothelium and resulted in > 90% clearance of a uropathogenic P. aeruginosa clinical strain in vivo.Attorney ref 340541: 12-25 WO

[0046] In an embodiment, a nanogel-CPP conjugate was synthesized compatible with gentamicin and its efficacy in treating a clinical isolate of an intracellular bacterial pathogen in a murine in vivo UTI model was characterized. As a result, the localized, intravesical I-VESIC delivery of antibiotic gentamicin to the site of IBCs within the urothelium was enabled. Gentamicin was chosen for its broad-spectrum antibiotic activity against P. aeruginosa and its widely reported impermeability to urothelial plasma membranes. This invention is applicable to targeted therapies for UTIs and other bladder disorders.

[0047] In an embodiment, extensive in vitro and in vivo data indicates that the novel, cellcompatible HTA nanogel conjugated with the CGKRK (SEQ ID NO:1) CPP can be loaded with GmGEN to successfully penetrate the urothelium and target uropathogenic P. aeruginosa for eradication from the murine bladder, highlighting it as a potential nanogelbased drug-delivery approach to treat UTIs. In an embodiment, more GPPs conjugated on the nanogel, different CPPs, or nanogels containing biofilm- disrupting molecules or antimicrobial peptides additionally enable targeted UTI treatments.

[0048] EXAMPLES

[0049] Example 1 - Nanoqels containing antibiotics

[0050] Nanogel Synthesis and CPP Conjugation

[0051] Following the protocol from a previously studied nanogel, 2-hydroxyethyl acrylate, tetraethyl dimethacrylate, and acrylic acid monomers (HTA) in 4X solvent ratio at ~70% double-bond conversion (HTA) was synthesized for this study via a one-pot, solution-based, free-radical polymerization with a thermal initiator

[0015] , HTA and CPP (synthesized by GenScript, Piscataway, NJ, USA) (3.8 x w3wt.%) were covalently linked with the residual acrylates on the HTA (1.3 x 1 o-3CPP unit per HTA) via thiol-acrylate Michael addition reaction as previously done by using 100 pL of trimethylamine as the catalyst in phosphate-buffered saline at pH ~7 (PBS) for 16 h while stirring

[0016] , Afterward, any residual acrylate functionality present was reacted with 2-mercaptoethanol in excess, then dialyzed (MWCO 10 kDa, cellulose membrane) against deionized water and lyophilized.

[0052] HTA-CPP Nanogel Characterization

[0053] The HTA molecular weight was determined by gel permeation chromatography (Viscoteck-270) with tetrahydrofuran (0.35 pL / min) as the mobile phase

[0015] , Additionally, the HTA-CPP size and zeta potential of HTA and HTA-CPP (0.5 mg / mL in PBS) wereAttorney ref 340541: 12-25 WOdetermined by dynamic light scattering using Zetasizer NanoZS (ZEN 3600, Malvern, Germany).

[0054] GEN Loading into HTA and HTA-CPP Nanogels

[0055] To load GEN (Chem-lmpex International; Wood Dale, IL) into HTA and HTA-CPP, a 20% (w / v) of each at 40 mg / mL GEN solution dispersed in PBS was mixed overnight. For loading HTA-CPP with tritiated GEN (GEN*), a 20% (w / v) of HTA-CPP was incubated in 100 pCi / mL of GEN* instead. The GEN-loaded HTA-CPP and HTA nanogels (HTA-CPP+GEN, HTA+GEN, and HTA-CPP+GEN* respectively) were isolated and dried from the free antibiotic after two 1 mL Milli-Q washes within a centrifugal filter (MWCO 10 kDa, polyether sulfone membrane). The filtrate in the collection tube was kept.

[0056] Loading Capacity and Encapsulation Efficiency

[0057] Following a published protocol, the GEN was collected as the filtrate was fluorescently derivatized with o-phthalaldehyde and quantified against a standard curve

[0018] , Fluorescence measurements (excitation (Aex) = 340 nm; emission (Aem) = 455 nm) were done on the Synergy™ 4 multi-mode microplate reader. The drug loading capacity and encapsulation efficiency of GEN in HTA-CPP+GEN and HTA+GEN were calculated with two equations where a is the total mass of GEN introduced to the nanogel for loading, p is the quantified mass of GEN that remained unloaded, and o is the total mass of nanogel used for drug loading

[0019] :

[0058] Equation 1 :oc - fLoading Capacity (%) = - x 100er

[0059] Equation 2:100

[0060] GEN Release Kinetics

[0061] GEN release from the HTA-CPP+GEN and HTA+GEN was monitored via dialysis

[0020] , Briefly, GEN release was monitored from a dialysis tube (MWCO 10 kDa, cellulose membrane) at a volume of 400 pL with a concentration of 1 mg / mL of the total weight of the HTA-CPP+GEN and HTA+GEN within a 40 mL PBS reservoir at 37°C with stirring. At 4-, 6-, 8-, 12-, 24-, and 96-h, samples from the reservoir were collected. The total sample volume removed by the four-hour timepoint was replaced with new PBS. GEN was quantified using the o-phthalaldehyde derivatization method described previously

[0018] ,Attorney ref 340541: 12-25 WO

[0062] Cell Viability by MTT Assay

[0063] A human carcinoma urothelial cell line was used in this study (ATCC® HTB-9). The HTB-9 cells were grown with Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v / v) fetal bovine serum. Cells were maintained at 37°C with 5% CO2until they reached ~70% confluency and were trypsinized (0.25% v / v trypsin) and subcultured.

[0064] The HTB-9 cells were seeded ~1 x 104cells per well and grown to confluency (> 90%) in a 96-well plate with complete RPMI medium. The filter-sterilized (0.22 pm) HTA-CPP were introduced to the cells in complete RPMI medium at increasing concentrations (0 pg - 10,000 pg) and incubated at 37°C with 5% CO2for 24 h. The introduction of 1% (v / v) Triton X-100 was used as a positive control. The negative control was cells without the presence of HTA-CPP (0 pg). Cell viability was determined by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay

[0021] , Briefly, the cells were washed three times with Dulbecco's PBS, supplemented with calcium and magnesium, followed by adding 0.5 mg / mL of the tetrazolium dye in RMPI medium and incubating for 1 h at 37°C with 5% CO2. An equal volume of RMPI medium was added with acidified isopropanol (4 mM HCI, 1% (v / v) Triton X-100) and incubated for 15 min on the orbital shaker. The absorbance was measured at 570 nm.

[0065] GEN* Uptake in Human Urothelial Cells

[0066] The HTA-CPP+GEN* was resolubilized in 50% (v / v) dimethyl sulfoxide in Dulbecco's Modified Eagle Medium to a final volume of 300 pL. Radioactivity of GEN* was measured in counts-per-minute using a scintillation counter. HTA-CPP+GEN* was added to HTB-9 cells to an approximate concentration of at least 1 pCi / mL for a total of 0.3 pCi per well. Concurrently, free GEN* was added to cells at a concentration normalized to counts-per-minutes of the HTA-CPP+GEN* to a concentration of approximately 0.7 pCi / mL. At 4 h, media from two samples was collected and placed into 5 mL of scintillation fluid to confirm equal treatment levels in terms of total radioactivity. Cells were washed one time with 1 mL of PBS with 12.5 pg / mL cold GEN. The cells were trypsinized (0.25% v / v trypsin) at 37°C for 5 min. Trypsinized cells were collected by centrifugation at 4,000 RPM for 5 min. The cell pellet was resuspended in 300 pL PBS and then added to 5 mL of scintillation fluid. T ritium counts were averaged over one minute for each sample. Counts were corrected for background.

[0067] Bacterial Strain, Plasmids, Media, and Culture ConditionsAttorney ref 340541: 12-25 WO

[0068] The uropathogenic P. aeruginosa clinical strain, UR34, was obtained from Feinberg School of Medicine in Northwestern University. The plasmid pMRP-1 was introduced into UR34 by electroporation to create UR34-GFP

[0022] , UR34-GFP growth medium was Lysogeny Broth (LB; 10 g / L casein peptone, 5 g / L yeast extract, and 5 g / L sodium chloride).

[0069] For the acute UTI model, UR34-GFP were grown statically in 2 mL of LB for 10 h at 37°C. The bacteria were collected by centrifugation from the broth and resuspended in PBS to ~ 1 x 109colony-forming-unit (CFU) per milliliter, then 50 pL (~1 - 5 x 1 o7CFU) was used to infect each mouse.

[0070] Antimicrobial Susceptibility

[0071] UR34-GFP were grown for 10 h statically at 37°C in LB. The culture was serially diluted to 105CFU / mL using GEN concentrations of 6.25-, 12.5-, 25-, 50-, 100-, and 200-pg / mL followed by incubation for 24 h at 37°C. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of antibiotic that no bacterial growth was visibly observed. From the tubes with no visible growth, 100 pL was spread onto LB agar plates and incubated overnight at 37°C. Plates were checked for growth and the lowest concentration of antibiotic that prevented 100% of bacterial growth was determined as the minimum bactericidal concentration (MBC).

[0072] Mice and l-VESIC Delivery Method in Murine Bladder

[0073] Adult male and female mice (C57BL / 68- 10 weeks old) were used in the study, and the method of delivery into the bladder was by the l-VESIC with a 24G x %” BD Insyte™ Autoguard™ Shield IV catheter under anesthesia and warming pad.

[0074] Histology and Evaluation of HTA-CPP Treated Murine Bladder

[0075] Three groups of uninfected mice (N = 3) were treated with HTA-CPP (250 pg in 50 pL PBS) twice a day by l-VESIC for one day (Group 1), two days (Group 2) or untreated (Group 3; control). All groups were euthanized, and the bladders were harvested, fixed with 4% paraformaldehyde, and further processed for myeloperoxidase (MPO) and F4-80 immunohistochemistry (IHC) staining, by the Gates Center Histology Core on the Anschutz Medical Campus. The sections were blinded, and then graded by a histopathologist using the following score scale for the relative number range of MPO and F4-80 IHC stained cells per 200x field quadrant and corresponding classfication: 0-2 cells, score = 0 (none); 3-10 cells, score = 1 (minor amounts); 11-25 cells, score = 2 (moderate amounts); 26-50 cells, score = 3 (extreme amounts).Attorney ref 340541: 12-25 WO

[0076] Nanogel Tracking in Murine Bladders Using Labelled Drug Mimic Delivery

[0077] Murine bladders were treated with HTA-CPP loaded with rhodamine B (25 pg in 50 pL PBS) as a drug mimetic delivered l-VESIC. The mice were kept under anesthesia for 15 min. Then, the bladders were emptied and washed three times via l-VESIC with 50 pL of PBS. At 15 min and 24 h post-wash, the bladders were harvested, fixed, and sectioned. Samples were dyed with Hoechst (1 pg / mL) for 15 min followed by imaging on the Zeiss Axio Observer 5 epifluorescent microscope equipped with X-Cite 200DC for the excitation and emission wavelengths with filter set for rhodamine B (Aex= 558 nm; Aem = 575 nm) and Hoechst (Aex= 353 nm; Aem= 465 nm).

[0078] Murine Bladder Infection

[0079] For the acute UTI model, mice were inoculated with 50 pL of 107CFU UR34-GFP by l-VESIC and were kept under anesthesia for 30 min, classified as zero days-post-infection (0 dpi). At 1 dpi and 2 dpi, urine samples were collected, serially diluted in PBS, and plated on LB agar for quantifying bacteriuria. Each day after the bladders were emptied, mice were treated twice per day for two days with 50 pL of 1 mg of GEN delivered subcutaneous (Sub-Q), 50pL of 1 mg GEN delivered l-VESIC via HTA+GEN or HTA-CPP+GEN or left untreated. Mice that were l-VESIC-treated were kept under anesthesia for 30 min before waking and allowed to void. At 3 dpi, the bladders and kidneys were aseptically harvested and homogenized (0.025% Triton X-100 in PBS) for bacterial titration on agar plates.

[0080] Murine Bladder Infected P. aeruginosa IBCs Imaging

[0081] Three additional infected mice were maintained under standard lodging conditions until 10 dpi when the bladders were aseptically harvested, rinsed and spread open on a microscope slide to be visualized. Intracellular imaging and confirmation of P. aeruginosa in the murine bladder was obtained using SlideBook6.0 software on a Zeiss Axioplan II digital microscope (Intelligent Imaging Incorporated, Denver, CO, USA) with the FITC channel.

[0082] Statistical Analysis

[0083] Data analysis was performed using GraphPad Prism 8 for Windows (Version 8.4.0).

[0084] Results

[0085] Previous work has shown that a nanogel composed of 2-hydroxyethyl acrylate and urethane dimethacrylate (i.e. , HAUD-nanogel) conjugated with the CGKRK (SEQ ID NO:1)Attorney ref 340541: 12-25 WOCPP had the ability to cross the stratified urothelium plasma membranes

[0016] , CGKRK (SEQ ID NO:1) is a small peptide, which, like other CPPs that are cationic, mediates cellular internalization and has been extensively used in nanoparticle drug delivery [23, 24], However, the HAUD-nanogels were more hydrophobic and would not allow the hydrophilic antibiotic GEN to be loaded. Therefore, a different nanogel design with an increased hydrophilicity to facilitate higher GEN loading was needed. Towards this end, the water-dispersible HTA nanogel, which has been shown to load hydrophilic small-molecules with high efficiency due to the high content of hydroxyl and carboxylic acid groups for hydrogen bonding and dipole-dipole interaction, was modified via a facile one-step, solution-based, free-radical polymerization protocol using 2-hydroxyethyl acrylate, tetraethyl dimethacrylate, and acrylic acid monomers (Figure 1)

[0015] , The particle size of the HTA-CPP in PBS was determined to be 97.3 nm ± 0.5 nm in radius and a molecular weight of ~20 kDa. There was no significant impact on the HTA anionic zeta potential, which changed from -7.4 mV ± 1.8 mV to -6.4 mV ± 0.8 mV (p = 0.43) after conjugating the surface with the cationic CPP.

[0086] While GEN is highly efficient in eradicating extracellular P. aeruginosa, it is reportedly impermeable to urothelial plasma membranes [25, 26], It was hypothesized that by loading the GEN within HTA-CPP (i.e., HTA-CPP+GEN), intracellular delivery of GEN could be enhanced because of the hydrogen bonding interaction between the hydroxyl, carboxylic acid, and amine groups found between GEN and HTA nanogel. To determine the feasibility of delivering HTA+GEN and HTA-CPP+GEN into infected cells, the nanogels’ encapsulation efficiency and loading capacity were assessed. The encapsulation efficiency for the HTA+GEN and HTA-CPP+GEN was 66.0% ± 0.1% and 60.0% ± 8.6% respectively. The loading capacity of GEN for HTA+GEN and HTA-CPP+GEN were similar at 13.2% ± 0.1% and 12.0% ± 1.7%, respectively. While the primary goal was to deliver HTA-CPP+GEN intracellularly, it was important to determine any interference caused by the CPP on the GEN released from the drug vehicle. Therefore, the GEN release kinetics from the HTA+GEN and HTA-CPP+GEN were assessed. In general, both the HTA+GEN and HTA-CPP+GEN exhibited a similar burst release profile at early time points and reached a maximum at 4 h (Figure 2). Near maximum drug release from both HTA+GEN and HTA-CPP+GEN was sustained up to 8 h with a steady decline from 12 h - 96 h. These observations provided the initial kinetic parameters for delivering GEN to treat uropathogenic P. aeruginosa infected murine bladders in vivo.

[0087] The HTA-CPP cytotoxicity to the HTB-9 cell line after 24 h exposure was assessed. Up to 1 mg HTA-CPP had no significant effect on the cell’s metabolism when compared to untreated cells (p > 0.05). In contrast, 10 mg HTA-CPP decreased the cellular metabolicAttorney ref 340541: 12-25 WOactivity significantly to ~19% compared to the untreated cells (p < 0.0001 ; Figure 3). These data indicate that there is a large safe dosage range for HTA-CPP before cellular toxicity was reached in vitro.

[0088] Radioactive GEN* was used to quantify the amounts of GEN* delivered as a free drug or delivered from HTA-CPP into HTB-9 cells in 4 h. Approximately 36% more GEN* was delivered by HTA-CPP than GEN* exogenously added to the cell culture medium (p < 0.01, Figure 4). These data indicate that while free GEN* is absorbed by HTB-9 cells from the culture medium, more intracellular GEN* was delivered by the HTA-CPP.

[0089] The MIC and MBC of GEN for P. aeruginosa UR34-GFP for GEN were determined to estimate the GEN amount required for delivery by HTA-CPP. The MIC was 12.5 pg / mL and the MBC was 25 pg / mL. However, studies that have used GEN for systemic delivery (i.e., subcutaneous or tail-vein injection) for treating various types of infection in rodent models, including UTIs, in vivo with P. aeruginosa had to use a therapeutic dosage in a range of 2.5 mg / kg - 80 mg / kg, which was significantly greater than the predetermined MIC and MBC in vitro [27-29], Therefore, in order to examine the efficacy of delivering a GEN dosage l-VESIC via HTA-CPP, 1000 pg GEN was delivered into the murine bladder (i.e., ~43 mg / kg) via Sub-Q or HTA-CPP that is within the 2.5 - 80 mg / kg dosage that is needed for a systemic therapeutic effect in the in vivo murine acute UTI model.

[0090] Before transitioning immediately into an in vivo murine infection model, it was determined whether the antibiotic-free HTA-CPP induced an inflammatory response in healthy murine bladders. Microscopic evaluation of H&E-stained bladder sections that were untreated and treated with HTA-CPP for 24 h and 48 h (Figure 5, panels A - C) showed evidence of minor edema in the lamina propria from each treated groups, and there was also a slight increase in urothelium thickness and sloughed cells in the murine bladder lumen. Yet, there was no sign of cell lysis. Furthermore, IHC showed there was no significant difference on the presence of murine macrophages F4-80 maker between control and HTA-CPP treated bladders at 24 h and 48h, yet there was a slight significant difference for the presence neutrophil MPO marker between groups (p = 0.045), indicating that the HTA-CPP overall has a safe usage with minimal side effects (Table 1 ; Figures 8 - 9).

[0091] Table 1Attorney ref 340541: 12-25 WO

[0092] It was previously demonstrated the penetration and delivery of rhodamine B into healthy murine bladders using HAUD-nanogel conjugated to the CGKRK (SEQ ID NO:1) CPP; however, HAUD-nanogel uptake required focal mechanical bladder injury

[0016] , Therefore, HTA-CPP loaded with rhodamine B was delivered to infected murine bladders without mechanical injury to determine whether HTA-CPP can penetrate infected murine bladder epithelium. Examination by ex vivo fluorescent imaging was performed on murine bladders untreated (control, Figure 5, panel D) and post-treated by l-VESIC delivery of the rhodamine B loaded HTA-CPP at 15 min (Figure 5, panel E) and 24 h (Figure 5, panel F). Rhodamine B was intracellular at 15 min (Figure 5, panel E) at higher relative fluorescence than background level (Figure 5, panel D) and reduced to near-background levels by 24 h (Figure 5, panel F), indicating that the HTA-CPP delivery system can be used to treat P. aeruginosa-infected murine bladders without inducing mechanical injury. These results also indicate that the rhodamine B was mostly cleared from the infected murine bladders within 24 h.Attorney ref 340541: 12-25 WO

[0093] Murine bladders were infected with the uropathogenic P. aeruginosa clinical isolate UR34 to determine if the HTA-CPP+GEN could be used on a CAUTI pathogen. Evidence is available that P. aeruginosa can establish IBCs in a murine UTI model but that study was conducted with a laboratory P. aeruginosa strain [5], To confirm that the clinical P. aeruginosa isolate UR34 was able to infect the bladders of mice, a plasmid encoding GFP was introduced into the isolate, mice were infected and urinary bladders collected to visualize the location of the bacteria. Intracellular P. aeruginosa UR34 were visualized by confocal microscopy residing within the murine bladder epithelium (Figure 6).

[0094] The infected mice were treated with the same GEN dose (1 mg / mL) to compare the efficacy of HTA, HTA-CPP (l-VESIC)-delivered GEN to the subcutaneously-delivered treatment. Determination of bacteriuria is the gold standard used in the clinic for UTI diagnosis. The urines from these treatment groups were collected at 1 and 2 dpi and there was no significant difference in bacterial loads between the treatment groups at these timepoints. However, the UR34 bacterial loads from HTA-CPP+GEN-treated urines from 2 dpi trended lower in comparison to the untreated infected mice urine (Figure 10, panels A and B). Moreover, the bacteria were not detected in -80% of the kidneys isolated from the 3 dpi HTA-CPP+GEN treatment group (Figure 10, panel C).

[0095] In order to determine if HTA-CPP+GEN was more effective at delivering GEN to the murine bladders than systemic (subcutaneous GEN) or no treatment, CFUs from these groups’ bladders were quantified at 3 dpi. Comparison of the CFUs obtained from the bladders of untreated infected mice to mice treated with GEN subcutaneously showed clearance of the bacterial load from four subcutaneously antibiotic treated mice, while no mice in the untreated group cleared the infection. In contrast, there was a statistically significant difference between the untreated mice and mice treated with HTA-CPP+GEN (p < 0.001, Figure 7, panel A). In order to determine if HTA-CPP+GEN was more effective at delivering GEN to the murine bladders than HTA without the CPP, infected mice were treated with HTA+GEN. There was significant difference between mice treated with HTA-CPP+GEN and HTA+GEN (p < 0.01), and 12 of the 16 mice treated with HTA-CPP+GEN cleared the P. aeruginosa UR34 from their bladders as opposed to 4 of the 16 mice treated with HTA+GEN (Figure 7, panel B). These results indicate that HTA-CPP+GEN was most efficacious in eliminating intracellular P. aeruginosa.

[0096] DiscussionAttorney ref 340541: 12-25 WO

[0097] This study was initiated to develop a targeted approach for treating UTIs by enabling the localized, l-VESIC delivery of an antibiotic to the site of IBCs within the urothelium. GEN was chosen for its broad-spectrum antibiotic activity against P. aeruginosa and its widely reported impermeability to urothelial plasma membranes. There have been a limited number of studies examining the efficacy of nanogels conjugated with a CPP and / or nanogels carrying antibiotics for the treatment of UTIs, including CAUTI [9-11], A novel nanogel-CPP conjugate was synthesized compatible with GEN and characterized its efficacy in treating a clinical isolate of an intracellular bacterial pathogen in a murine in vivo UTI model.

[0098] The average HTA-CPP radius was formulated at -100 nm, within the average range of 10 - 200 nm for nanogels that are typically evaluated for drug delivery systems

[0030] , This scale ensured that the nanogels were positioned for cellular uptake. Upon determining the GEN release kinetics, the GEN loading capacity for HTA-CPP (loading capacity = -12%) was comparable to other GEN-loaded nanogel-based drug delivery systems that span between 6% - 32% [31 , 32], Therefore, the loading capacities attained were deemed sufficient for this study since the final GEN amount delivered can be adjusted to attain thereapuetic doses.

[0099] An advantage of this nanogel synthesis protocol is the ease of mitigating the toxicity that arises from nano-scale particles that depends on multiple physicochemical properties

[0033] , While a combination of factors contribute to toxicity, toxicity increases as the radius decreases below -50 nm [34, 35], Other factors that determine nanogel toxicity are 1) chemical composition, such as the organic and inorganic building blocks that are used to construct the nanogel, and 2) zeta potential, where studies have shown that maintaining the zeta potential between -10 mV and +10 mV is advantageous since it does not electrostatically attract other charged biomolecules due to a relatively ‘neutral’ charge [13, 35, 36], The monomers for HTA synthesis were chosen fortheir low toxicity and hydrophilicity [15, 37-39], Moreover, the anionic HTA differs from other nanoparticle-based delivery approaches for treating UTIs that are relatively cationic (range +10 mV - +20 mV) [40-43], The advantage of using cationic nanoparticles is that it can electrostatically interact with the anionic surfaces of bacteria and induce intracellular uptake, produce reactive oxygen species, disrupt bacterial membranes, deplete ATP, and impact gene and protein regulation [44-47], However, delivering considerable amounts of these cationic nanoparticles also disrupts eukaryotic mitochondria and lysosome plasma-membranes which limits the usable concentration of nanogels

[0048] , These results support the hypothesis that the HTA’sAttorney ref 340541: 12-25 WOintrinsic anionic properties leads to lower cytotoxicity and better cellular uptake into the urothelial cells via the conjugated cationic CPP without significant impact, thereby permitting higher drug concentrations for treatment.

[0100] The ideal drug release kinetics for any drug delivery system is subjective and depends on multiple factors such as the disease or infection under study, target location for delivery, and the nature of the drug. For example, a burst release is a preferred mechanism to treat acute infections, while a sustained release of a drug is preferred in conditions that conventionally require frequent doses. The GEN exhibited a burst release from HTA-CPP, which is advantageous for achieiving a high dose environment, thereby killing planktonic pathogens found in the bladder lumen before it can reestablish IBCs. However, rapid drug release into the bladder prior to HTA-CPP cellular internalization is at risk for washout by urinary flow and diluting the intracellular drug availability

[0049] , The rapid release kinetics may explain why there was only slight statistical increase in intracellular GEN delivered by HTA-CPP in vitro versus the free GEN. A rapid release coupled with a controlled release of the drug would be more desirable for maintaining a higher intracellular GEN concentration over time, as shown by the sustained GEN release activity from the polymethysilsesquioxane capsules over days in treating another uropathogen E. faecalis [9], Futhermore, controlled drug release would prevent the drug resistance evolution from having low dose exposure overtime

[0050] , Within the current HTA, strategies for controlled GEN release, such as using specific environmental triggers (e.g., light, pH, thermal, and enzymatic) to facilitate drug release from the nanogel, are feasible and are currently being investigated [15, 51],

[0101] The HTA-CPP approach towards treating UTIs is advantageous due to its targeted, local delivery and ability to penetrate the urothelium. Current standards for antibiotic therapeutics against UTIs are delivered systemically via oral or intravenous administration routes

[0052] , However, these practices require high doses to reach therapeutic concentrations in vivo because the bacteria reside within the urothelium. Consequently, high antibiotic doses increase harmful side effects on other organs such as the kidneys

[0053] , Alternatively, local drug delivery increases biodistribution at the target organ and abrogates side effects

[0011] , Currently, bladder instillations with GEN, which can decrease antibiotic resistance and symptomatic UTI episodes, are performed on patients with refractory UTIs when other first-line UTI therapies fail

[0049] , Yet, drug instillation into the bladder does not clear the infection, in part due to the bladder permeability barrier that prevents drug penetration into the underlying cells

[0050] , In the murine model, the HTA-CPP delivered the GEN payload l-VESIC to the bladder and cleared a majority of the P. aeruginosa infected murine bladders compared to other similar studies using GEN [28, 29, 54], These resultsAttorney ref 340541: 12-25 WOprovide the potential for using the HTA-CPP as a drug-carrier vehicle for bladder instillations to treat UTIs in the clinic with the advantage of delivering less drug directly to the infection site and increasing drug biodistribution to the urothelium. Furthermore, the HTA-CPP are stable at room temperature and can be easily adapted for similar delivery in an office setting. However, systemic antibiotics cause side effects to patients and the emergence of antimicrobial resistance in uropathogenic bacteria.

[0102] GEN is a broad-spectrum antibiotic that is typically used for moderate to severe infections, particularly against Gram-negative organisms and is considered to be a nonmembrane permeable antibiotic [25, 26], However, several studies have used different methods to make GEN permeable to bladder epithelia. For instance, Gomarasca et al.

[0055] used a CPP-conjugated GEN treatment to kill intracellular E. coli K1 RS218 and reported a significant reduction of the intracellular bacteria. Kim et al.

[0056] demonstrated GEN can enter into host cells in vitro, presumably through pinocyotisis, leading to inaccurate numbers of intracellular CFU, thereby indicating significant variations on the outcome. Additionally, Blango and Mulvey

[0057] used GEN as one of many antibiotics to treat against the clinical isolate bacterial strain, UTI89-GFP. The study demonstrated that GEN reduced, but did not eliminate, the uropathogenic Escherichia coli CFUs in biofilm and within host urothelial cells; the combination of biofilm formation physiology and the urothelial cell barrier were cited as likely causes for uropathogenic Escherichia coli resistance to GEN treatment. These causes may be applicable here for the pathogensis of UR34 where the IBCs established as biofilms in the urothelial cell’s cytoplasm and could have a subset of pathogens establishing longterm reservoirs in endosomal-lysosomal vesicles

[0058] , Additional cell-compartment targeting molecules like lysosomol sorting peptides have shown promise for improving target drug delivery to accomplish uropathogen eradication while reducing antimicrobial resistance and side effects from drugs delivered systemically

[0059] , In the future, more GPP conjugated on the nanogel, different CPPs, or nanogels containing biofilm-disrupting molecules or antimicrobial peptides could be explored as alternatives for improvement.

[0103] Overall, these findings demonstrate that the novel, cell-compatible HTA nanogel conjugated with the CGKRK (SEQ ID NO:1) CPP can be loaded with GEN to successfully penetrate the urothelium and target uropathogenic P. aeruginosa for eradication from the murine bladder, highlighting it as a potential nanogel-based drug delivery approach to treat UTIs.

[0104] Supplemental Information - Method: Nanogel SynthesisAttorney ref 340541: 12-25 WO

[0105] 2-hydroxyethyl acrylate, tetraethyl dimethacrylate, and acrylic acid monomers (60:20:20 molar ratios respectively) were reacted with each other to form a nanogel. The monomers were mixed in with athermal initiator 2, 2'-azobis(2-methylpropionitrile) (1 wt.%), a chain-transfer agent 2-mercaptoethanol (20 mol%) and the solvent methyl ethyl ketone (200 g / L) while stirring at 85°C at 200 RPM for 45 min. Once the reaction was ~70% completed, as monitored via double-bond conversion of the acrylate / methacrylate groups at 1639 cm1on Fourier-transform infrared spectroscopy, the nanogel was precipitated into a ten-fold excess of hexane and the residual solvent was removed via rotary evaporator.

[0106] Example 2 - Nanogels containing estradiol

[0107] Urinary tract infections (UTIs) are among one of the most common bacterial infections that disproportionately impact women and children. Among women, the recurrent occurrence of UTIs or rUTIs occurs in 1 out of 4 women where typical symptoms can include suprapubic pain, increased urgency and frequency of urination, and general discomfort. In postmenopausal women, the lower levels of estradiol are seen to make them more susceptible to rUTIs as estradiol has been shown to play a key role in boosting the bladder’s innate defenses by enhancing epithelial structure and barrier via the increased expression of genes associated with cell proliferation and extracellular matrix protein synthesis.

[0108] Clinical results show that low estrogen levels with postmenopausal women can cause the urethra to become thin, making it more prone to infections [60, 61], This is because within the bladder, estradiol can bind to estrogen receptor alpha leading to transcriptional regulation of genes involved in the control of cell proliferation and survival

[0062] , Uropathogenic E coli (UPEC) can form intracellular biofilm communities (IBCs) that enable them to evade antibiotic and immune clearance

[0063] , While vaginal delivery of estradiol via gels, inserts, and rings have been explored, the means of effectively delivering estradiol directly to the urothelium have been limited. Additionally, although selfadministered topical vaginal estradiol creams have been used to treat UTIs in the past, antibiotic regimens and creams are inefficient in penetrating the stratified urothelium where the IBCs reside

[0064] ,

[0109] This example examines the direct delivery of estradiol to the bladder using nanogels containing cell-penetrating peptides (CPPs) as a method to improve the bladder’s ability to clear uropathogenic E.coli and take the first step towards developing effective therapies for high-risk patients with recurring UTIs.Attorney ref 340541: 12-25 WO

[0110] In this example, nanogels were chosen as the carrier for the intravesicular delivery of estradiol. Nanogels, which are polymeric 3-D networks, have the ability to swell and collapse, and have been used extensively as drug delivery vehicles in recent years. The ability to swell in various aqueous solvent provides the opportunity to swell in a smallmolecules within the nanogel, which can then be released at an appropriate point in time. As nanogels can be tailored to be hydrophilic, hydrophobic or amphiphilic based on the monomers chosen to create the nanogel, a wide array of small-molecules with different functionalities can be encapsulated within system. Nanogels can also be functionalized on the surface with different ligands or targeting moieties such as CCPs, making them targeted drug delivery systems. In this example, a novel amphiphilic nanogel conjugated with a cell penetrating peptide that can target the nanogel to the urothelium was synthesized to deliver estradiol intravesically in the bladder.

[0111] Materials and Methods

[0112] Polyethylene glycol) methacrylate (HEMA10), Polyethylene gylcol) dimethacrylate (PEGDMA750), Stearyl Acrylate (SA), 2,2’-azobis(2-methylpropionitrile) (AIBN) 98% and triethylameine 99% were all obtained from Sigma-Aldrich. Cys-Gly-Lys-Arg-Lys cell penetrating peptide (CPP) synthesized from GenScript and all solvents used were obtained from Fisher (see Figure 11).

[0113] Synthesis of Nanogel (NG)

[0114] SA: HEMA10:PEGDMA750 in a 25:55:20 molar ratio was synthesized herein denoted as SHP NG. 1wt% of the thermal initiator, AIBN, was added and stirred at 200 RPM (80°C) using 4x methyl ethyl ketone (MEK) as the solvent and stopped at 70% conversion. The double bond conversion of the acrylate groups was monitored via FTIR spectroscopy (mid-IR, 815 cm-1). The nanogel was then precipitated in a 10-fold excess of hexanes and after decanting, the remaining gel was then re-suspended in dichloromethane. Any residual solvent was then was removed completely via a rotary evaporator until a gel-like NG was achieved. Afterwards, the nanogel (NG) was resuspended in phosphate-buffered saline (1x PBS) where 3.8 x 10-3wt.% of the CPP which was covalently linked with the residual acrylate groups on the NG using triethylamine as a catalyst for 16 hours. Subsequently, the nanogel was then dialyzed with a cellulose membrane dialysis bag with a molecular weight cut off (MWCO) of 16kDa against Milli-Q water. After two water changes, the contents were then dried at 37°C under vacuum until the final product was isolated.Attorney ref 340541: 12-25 WO

[0115] The molecular weight and size of the resulting nanogel was analyzed via dynamic light scattering using a Zetasizer NanoZS (Zen 3600, Malvern, Germany) at room temperature with tetrahydrofuran as a solvent.

[0116] Encapsulating Estradiol in Nanogels

[0117] A 20mg / ml_ estradiol solution in 200 proof ethanol with 20% (w / v) of SHP-CPP was mixed overnight. The estradiol loaded nanogel system was isolated from the free estradiol with a centrifugal filter (MWCO 10 kDa, polyether sulfone membrane) at 4300 RPM. This was then followed by two 70% ethanol washes at two times the volume of the remaining in the concentrator. Final product was collected from the top filter and dried.

[0118] The filtrate was collected for indirectly quantifying the loading capacity and encapsulation efficiency using the following equations:100Total Estradiol Mass - Quantified PreeEstradiol~ - r- - --w; - » 100Tarry Estradiol Mass

[0119] Drug elution from Nanogel

[0120] Estradiol release from the SHP NG was monitored via dialysis in a 10ml_ 1x PBS reservoir at 24°C. 125pg of estradiol that was incapsulated in 1.1 mg of nanogel was resuspended in 1ml_ of 1x PBS and placed in a cellulose membrane dialysis bag. Samples from the reservoir were taken at predetermined points and replaced such that the volume was maintained at 10mL. On day four, the dialysis bag was placed in a fresh reservoir. Estradiol was detected by measuring absorbance of 279 and 288 nm light. The readings were then normalized relative to the theoretical maximum concentration of estradiol released given by that encapsulation efficiency calculated earlier.

[0121] Cytotoxicity

[0122] Human urothelial cells were grown into a 96-well plate with RMPI media until ~70% confluence was reached. Nanogel at concentrations between 0 - 50mg / mL in RMPI were prepared and sterilized with a 0.22pm filter. The media was then aspirated out before and washed with 1x PBS before the various concentrations of nanogel were introduced to the wells. 100pL of each concentration was transferred and incubated with the cells at 37°C with 7.5% CO2for 24 hours followed by MTT assay. Briefly, the nanogel and media solution was removed and the cells were washed three times with PBS. Fresh RMPI media wasAttorney ref 340541: 12-25 WOadded with the addition of 1 OpL of 5 mg / mL of tetrazolium dye followed by incubation for 4 h at 37°C with 7.5% CO2. This was followed with, 100pL of (20% sodium dodecyl sulfate + 50% dimethylformamide) solution in Milli-Q water was added and incubated overnight at 37°C. Absorbance measurement were done at 570 nm. Percent viability was then determined by comparison of the optical density of the extract cultures to the controls with cells only.

[0123] In Vivo Mouse Model

[0124] Ovariectomized C57J black mice were infected with 107 CFUs (Colony Forming Units) of UTI89 and let to rest for 3 days. After the 3 days, the mice were treated with 200 pgs of gentamicin subcutaneously once daily for 5 days. After the gentamicin treatment, the mice in the untreated group were left untouched until the harvest date. The nanogel-treated mice received estradiol nanogels (125 pg of estradiol in 1.1 mg of nanogel) dispersed in PBS intravascularly every 3 days for 2 weeks until the harvest date. Following the harvest, the bladders, spleen, and uteri were extracted from each mouse. The bladders were sectioned in half, homogenized, serially diluted, and plated on LB agar to quantify CFUs.

[0125] Nanogel Synthesis and Characterization

[0126] The SHP NG synthesis was monitored by following the degree of conversion of the acrylate groups in mid-FTIR until 70% conversion was achieved. The molecular weight (Mw) and hydrodynamic radius (Rh) of the SHP NG were measured at on the Zetasizer to be 166 kDa and 26.8 nm, respectively. The loading capacity and encapsulation efficiency was calculated to be 11.70% and 97.84% respectively.

[0127] Small Molecule Release

[0128] The ability for the SHP nanogel to curb the burst release kinetics and sustain the delivery of estradiol was studied over the course of 2 weeks. The nanogel networks were able to curb the burst release of estradiol as 35% of the total estradiol content was released in the first day, but up to 86% was released by day 12, showing that the nanogel can extend the delivery of a small molecule (see Figure 12).

[0129] SHP cytotoxicity and characterization

[0130] MTT assay (testing extractables and leachables) shows urothelial cell survival was unimpacted by SHP NG up to 1000 pg (see Figure 13).

[0131] In-vivo StudiesAttorney ref 340541: 12-25 WO

[0132] UPEC CFU / mg recovered from estradiol-treated mouse bladders were significantly lower showing a 93% fewer CFUs recovered on average than the untreated mouse bladder indicating that estradiol elicited an antibacterial response in the bladder (see Figure 14).

[0133] Discussion

[0134] Nanogels have been used in recent years as a drug delivery system due to its ability to easily be injected within the body and offer direct delivery of a drug to the desired area. The localized, targeted delivery of small-molecules have the advantages of higher bioavailability and less side- effects when compared to systemic delivery. In this example, amphiphilic nanogels tailored to encapsulate estradiol were synthesized and characterized in-vitro before being used in acute UTI mouse model. As estradiol has a hydrophobic backbone with hydrophilic moieties present only on the extremities of the molecule, the hydrophobic monomer stearyl acrylate was chosen. However, as loading estradiol within the nanogels and the intravesical delivery of estradiol-loaded nanogels require the nanogel to disperse in relatively hydrophilic solvent such as ethanol and / or phosphate buffered solutions, hydrophilic monomers HEMA10 and PEGDMA were also chosen to create the nanogel. Thus, the synthesized SHP nanogel, which was subsequently conjugated with the cell-penetrating peptide CGKRK, observed to have a molecular weight of 166kDa and hydrodynamic radius of 26.8nm, swelled and dispersed easily in ethanol and PBS. The added dialysis step for this nanogel insured than there were no residual monomers present in the system that could have a negative impact on the cytotoxicity.

[0135] The nanogel was then characterized for loading capacity and encapsulation efficiency. The loading capacity, which quantifies the amount of estradiol that can be encapsulated as a function of nanogel concentration was observed to be 11%. Although this percentage is not very high, the SHP nanogel is still able to deliver therapeutic amounts of estradiol and sustain the extended-release kinetics that is desired. On the other hand, the encapsulation efficiency was measured at 97%, indicating that the nanogel was efficient in containing high concentrations of estradiol within its network. Subsequently, monitoring the estradiol release from the nanogel showed that approximately 20% of the total estradiol loaded is released in the first six hours in a controlled manner, and the sustained release of estradiol was observed up to10 more days as shown in Figure 12.

[0136] By synthesizing an amphiphilic nanogel conjugated with CPPs (with specific examples having a molecular weight of 166 kDa, hydrodynamic radius (Rh) of 26.8nm, loading capacity of 11.70% and encapsulation efficiency of 97.84%), this example showsAttorney ref 340541: 12-25 WOthat nanogels can deliver therapeutic doses of estradiol intravesically in an acute rUTI mouse model. Intravesically delivered estradiol, in conjunction with subcutaneous gentamicin was able to achieve an average of 93% reduction in colony forming units when compared to the control.

[0137] Statements Regarding Incorporation by Reference and Variations

[0138] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0139] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the product, product components, methods steps set forth in the present description. As will be obvious to one of skill in the art, products and methods useful for the present invention can include a large number of optional composition and processing elements and steps.

[0140] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. The expression “of any of embodiments XX- YY” (wherein XX and YY refer to embodiment numbers) is intended to provide a multiple dependencies in theAttorney ref 340541: 12-25 WOalternative form, and in some embodiments is interchangeable with the expression “as in any one of embodiments XX- YY.”

[0141] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination.

[0142] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the embodiments herein.

[0143] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.

[0144] As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the said embodiment. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the embodiment. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.Attorney ref 340541: 12-25 WO

[0145] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by embodiments herein.

[0146] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the products and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.

[0147] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art.Attorney ref 340541: 12-25 WOREFERENCES

[0148] 1. Foxman, B„ Urinary Tract Infection Syndromes: Occurrence, Recurrence, Bacteriology, Risk Factors, and Disease Burden. Infectious Disease Clinics, 2014. 28(1): p.1-13.

[0149] 2. Ronald, A., The etiology of urinary tract infection: Traditional and emerging pathogens. Disease-a-Month, 2003. 49(2): p. 71-82.

[0150] 3. Foxman, B., Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. The American Journal of Medicine, 2002. 113(1, Supplement 1): p. 5-13.

[0151] 4. Foxman, B., The epidemiology of urinary tract infection. Nature Reviews Urology, 2010. 7(12): p. 653-660.

[0152] 5 Penaranda, C., N.M. Chumbier, and D.T. Hung, Dual transcriptional analysis reveals adaptation of host and pathogen to intracellular survival of Pseudomonas aeruginosa associated with urinary tract infection. PLOS Pathogens, 2021. 17(4): p. e1009534.

[0153] 6 Newman, J.W., R.V. Floyd, and J.L. Fothergill, The contribution of Pseudomonas aeruginosa virulence factors and host factors in the establishment of urinary tract infections. FEMS Microbiology Letters, 2017. 364(15): p. fnx124.

[0154] 7. Medina, M. and E. Castillo-Pino, An introduction to the epidemiology and burden of urinary tract infections. Therapeutic Advances in Urology, 2019. 11: p.1756287219832172.

[0155] 8. Hassett, D.J., et al., The Bactericidal Tandem Drug, AB569: Howto Eradicate Antibiotic-Resistant Biofilm Pseudomonas aeruginosa in Multiple Disease Settings Including Cystic Fibrosis, Burns / Wounds and Urinary Tract Infections. Frontiers in Microbiology, 2021.12.

[0156] 9. Labbaf, S., et al., An encapsulated drug delivery system for recalcitrant urinary tract infection. Journal of The Royal Society Interface, 2013. 10(89): p. 20130747.

[0157] 10. Gao, W., et al., Nanoparticle-based local antimicrobial drug delivery.Advanced Drug Delivery Reviews, 2018. 127: p. 46-57.

[0158] 11 Mittal, R., et al., Local drug delivery in the urinary tract: current challenges and opportunities. Journal of Drug Targeting, 2018. 26(8): p. 658-669.

[0159] 12 Soni, K.S., S.S. Desale, and T.K. Bronich, Nanogels: An overview of properties, biomedical applications and obstacles to clinical translation. Journal of Controlled Release, 2016. 240: p. 109-126.Attorney ref 340541: 12-25 WO

[0160] 13. Verma, A. and F. Stellacci, Effect of Surface Properties on Nanoparticle-Cell Interactions. Small, 2010. 6(1): p. 12-21.

[0161] 14. Zhang, L, et al., Interplay of Nanoparticle Rigidity and Its Translocation Ability through Cell Membrane. The Journal of Physical Chemistry B, 2019. 123(42): p. 8923-8930.

[0162] 15. Escobedo, H.D., J.W. Stansbury, and D.P. Nair, Photoreactive nanogels as versatile polymer networks with tunable in situ drug release kinetics. Journal of the Mechanical Behavior of Biomedical Materials, 2020. 108: p. 103755.

[0163] 16. Griffin, J.I., et al., Cell-penetrating peptide CGKRK mediates efficient and widespread targeting of bladder mucosa following focal injury. Nanomedicine:Nanotechnology, Biology and Medicine, 2017. 13(6): p. 1925-1932.

[0164] 17 Catania, R., et al., Study on Significance of Receptor Targeting in Killing of Intracellular Bacteria with Membrane-Impermeable Antibiotics. Advanced Therapeutics, 2021. 4(12): p. 2100168.

[0165] 18. Gubernator, J., Z. Drulis-Kawa, and A. Kozubek, A simply and sensitive fluorometric method for determination of gentamicin in liposomal suspensions. International Journal of Pharmaceutics, 2006. 327(1): p. 104-109.

[0166] 19. Ritsema, J.A.S., et al., Relationship between polarities of antibiotic and polymer matrix on nanoparticle formulations based on aliphatic polyesters. International Journal of Pharmaceutics, 2018. 548(2): p. 730-739.

[0167] 20. D’Souza, S., A Review of in Vitro Drug Release Test Methods for Nano-Sized Dosage Forms. Advances in Pharmaceutics, 2014. 2014(1): p. 304757.

[0168] 21. Postma, A., et al., Self-Polymerization of Dopamine as a Versatile and Robust Technique to Prepare Polymer Capsules. Chemistry of Materials, 2009. 21(14): p. 3042-3044.

[0169] 22. Diver, J.M., L.E. Bryan, and P.A. Sokol, Transformation of Pseudomonas aeruginosa by electroporation. Anal Biochem, 1990. 189(1): p. 75-9.

[0170] 23. Ruoslahti, E., Peptides as Targeting Elements and Tissue Penetration Devices for Nanoparticles. Advanced Materials, 2012. 24(28): p. 3747-3756.

[0171] 24. Hu, Q., et al., CGKRK-modified nanoparticles for dual-targeting drug delivery to tumor cells and angiogenic blood vessels. Biomaterials, 2013. 34(37): p. 9496-9508.Attorney ref 340541: 12-25 WO

[0172] 25. Rosenkrantz, B.E., J.R. Greco, J.G. Hoogerheide, and E.M. Oden, Gentamicin Sulfate, in Analytical Profiles of Drug Substances, K. Florey, Editor. 1981, Academic Press, p. 295-340.

[0173] 26. Tangy, F., et al., Mechanism of action of gentamicin components. European Journal of Biochemistry, 1985. 147(2): p. 381-386.

[0174] 27. Goering Richard, V., C. Sanders Christine, and W.E. Sanders, Comparison of 5-Episisomicin (Sch 22591), Gentamicin, Sisomicin, and Tobramycin in Treatment of Experimental Pseudomonas Infections in Mice. Antimicrobial Agents and Chemotherapy, 1978. 14(6): p. 824-828.

[0175] 28. Nicas, T.l. and L.E. Bryan, Relationship Between Gentamicin Susceptibility Criteria and Therapeutic Serum Levels for Pseudomonas aeruginosa in Mouse Infection Model. Antimicrobial Agents and Chemotherapy, 1978. 13(5): p. 796-801.

[0176] 29. LeBrun, M., et al., Effectiveness and Toxicity of Gentamicin in an Experimental Model of Pyelonephritis: Effect of the Time of Administration. Antimicrobial Agents and Chemotherapy, 1999. 43(5): p. 1020-1026.

[0177] 30. HKS, Y., A.H. NA, and A. GA, Nanogels as Novel Drug Delivery Systems - A Review. Journal of Pharmacy and Pharmaceutical Research, 2017. 1(1:5).

[0178] 31. Zabihian, A., et al., Preparation of Composite Nanogel Carrier for Gentamicin Delivery. Research in Pharmaceutical Sciences, 2012. 7(5).

[0179] 32. Zabihian, A., M. Salouti, and M. Hamidi, Factorial design analysis and optimisation of chitosan-based nanogels as controlled release system for gentamicin. IET Nanobiotechnology, 2018. 12(1): p. 12-17.

[0180] 33. Sayes, C.M. and D.B. Warheit, Characterization of nanomaterials for toxicity assessment. WIREs Nanomedicine and Nanobiotechnology, 2009. 1(6): p. 660-670.

[0181] 34. Oyewumi, M.O., A. Kumar, and Z. Cui, Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Review of Vaccines, 2010. 9(9): p. 1095-1107.

[0182] 35. Shang, L, K. Nienhaus, and G.U. Nienhaus, Engineered nanoparticles interacting with cells: size matters. Journal of Nanobiotechnology, 2014. 12(1): p. 5.

[0183] 36. Srivastava, V., D. Gusain, and Y.C. Sharma, Critical Review on the Toxicity of Some Widely Used Engineered Nanoparticles. Industrial & Engineering Chemistry Research, 2015. 54(24): p. 6209-6233.Attorney ref 340541: 12-25 WO

[0184] 37. Wilems, T.S., et al., Effects of free radical initiators on polyethylene glycol dimethacrylate hydrogel properties and biocompatibility. Journal of Biomedical Materials Research Part A, 2017. 105(11): p. 3059-3068.

[0185] 38. Siafaka, P.I., et al., Porous dressings of modified chitosan with poly(2-hydroxyethyl acrylate) for topical wound delivery of levo floxacin. Carbohydrate Polymers, 2016. 143: p. 90-99.

[0186] 39. Xu, M., et al., Improved In Vitro and In Vivo Biocompatibility of Graphene Oxide through Surface Modification: Poly(Acrylic Acid)-Functionalization is Superior to PEGylation. ACS Nano, 2016. 10(3): p. 3267-3281.

[0187] 40. Slavin, Y.N., J. Asnis, U.O. Hafeli, and H. Bach, Metal nanoparticles: understanding the mechanisms behind antibacterial activity. Journal of Nanobiotechnology, 2017. 15(1): p. 65.

[0188] 41. Divya, M., G.S. Kiran, S. Hassan, and J. Selvin, Biogenic synthesis and effect of silver nanoparticles (AgNPs) to combat catheter-related urinary tract infections.Biocatalysis and agricultural biotechnology, 2019. 18: p. 101037.

[0189] 42. Lopez-Carrizales, M., et al. In Vitro Synergism of Silver Nanoparticles with Antibiotics as an Alternative Treatment in Multiresistant L / ropathogens. Antibiotics, 2018. 7, DOI: 10.3390 / antibiotics7020050.

[0190] 43. Rodriguez-Serrano, C., et al., Biosynthesis of silver nanoparticles by Fusarium scirpi and its potential as antimicrobial agent against uropathogenic Escherichia coli biofilms. PLOS ONE, 2020. 15(3): p. e0230275.

[0191] 44. Naqvi, S.Z., et al., Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. I nt J Nanomedicine, 2013. 8: p. 3187-95.

[0192] 45. Payne, J.N., et al., Novel Synthesis of Kanamycin Conjugated Gold Nanoparticles with Potent Antibacterial Activity. Frontiers in Microbiology, 2016. 7.

[0193] 46. Morones-Ramirez, J.R., J.A. Winkler, C.S. Spina, and J. J. Collins, Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria. Science Translational Medicine, 2013. 5(190): p. 190ra81-190ra81.

[0194] 47. Wozniak-Budych, M.J., et al., Green synthesis of rifampicin-loaded copper nanoparticles with enhanced antimicrobial activity. Journal of Materials Science: Materials in Medicine, 2017. 28(3): p. 42.Attorney ref 340541: 12-25 WO

[0195] 48. Frohlich, E„ The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int J Nanomedicine, 2012. 7: p. 5577-91.

[0196] 49. Pichl, C.M., et al. Biomimickry of UPEC Cytoinvasion: A Novel Concept for Improved Drug Delivery in UTI. Pathogens, 2016. 5, DOI: 10.3390 / pathogens5010016.

[0197] 50. Tyagi, P., et al., Recent advances in intravesical drug / gene delivery. Mol Pharm, 2006. 3(4): p. 369-79.

[0198] 51. Ahmed, S., K. Alhareth, and N. Mignet, Advancement in nanogel formulations provides controlled drug release. International Journal of Pharmaceutics, 2020. 584: p. 119435.

[0199] 52. Ventola, C.L., The antibiotic resistance crisis: part 2: management strategies and new agents. Pt, 2015. 40(5): p. 344-52.

[0200] 53. Arora, D., et al., An update on polysaccharide-based nanomaterials for antimicrobial applications. Applied Microbiology and Biotechnology, 2016. 100(6): p. 2603-2615.

[0201] 54. Hopkins Walter, J., A. Gendron-Fitzpatrick, E. Balish, and T. Uehling David, Time Course and Host Responses to Escherichia coli Urinary Tract Infection in Genetically Distinct Mouse Strains. Infection and Immunity, 1998. 66(6): p. 2798-2802.

[0202] 55. Gomarasca, M., et al., Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens. Antimicrobial Agents and Chemotherapy, 2017.61(4): p. 10.1128 / aac.02545-16.

[0203] 56. Kim, J.-H., et al., Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci. Infection and Immunity, 2019. 87(5): p. 10.1128 / iai.00119-19.

[0204] 57. Blango Matthew, G. and A. Mulvey Matthew, Persistence of Uropathogenic Escherichia coli in the Face of Multiple Antibiotics. Antimicrobial Agents and Chemotherapy, 2010. 54(5): p. 1855-1863.

[0205] 58. Wang, C., et al., Atg16L1 deficiency confers protection from uropathogenic Escherichia coli infection in vivo. Proc Natl Acad Sci U SA, 2012. 109(27): p. 11008-13.

[0206] 59. Dekiwadia, C.D., A.C. Lawrie, and J.V. Fecondo, Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes. J Pept Sci, 2012. 18(8): p. 527-34.Attorney ref 340541: 12-25 WO

[0207] 60. Luthje P, Linden Hirschberg A, Brauner A., Estrogenic action on innate defense mechanisms in the urinary tract. Maturitas [Internet], 2014;77(1):32-6.

[0208] 61. Wawrysiuk S, Naber K, Rechberger T, Miotla P. Prevention and treatment of uncomplicated lower urinary tract infections in the era of increasing antimicrobial resistance — non-antibiotic approaches: a systemic review. Arch Gynecol Obstet [Internet], 2019;300(4):821-8.

[0209] 62. Yu X, Zhang X, Dhakal IB, Beggs M, Kadlubar S, Luo D. Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells. BMC Cancer. 2012;12(1): 29.

[0210] 63. Ronald A. The etiology of urinary tract infection: Traditional and emerging pathogens. Disease-a-Month. 2003;49(2):71-82.

[0211] 64. Mysorekar IU, Hultgren SJ. Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc Natl Acad Sci U SA.2006 ;103(38): 14170-5.

Claims

Attorney ref 340541: 12-25 WOCLAIMSWe claim:

1. A therapeutic composition comprising:a) a nanogel comprising a plurality of hydrogel particles having an average diameter from 1 nm to 1,000 nm, wherein each of the hydrogel particles comprise one or more crosslinked polymers;b) one or more cell-penetrating peptides (CPPs) conjugated to the hydrogel particles of the nanogel; andc) one or more therapeutic agents, wherein the one or more therapeutic agents comprises one or more antimicrobial agents, one or more steroid hormones, or combinations thereof, and wherein the one or more therapeutic agents are at least partially encapsulated by the nanogel.

2. The therapeutic composition of claim 1 , wherein the one or more CPPs comprise a peptide having the sequence CGKRK (SEQ ID NO: 1).

3. The therapeutic composition of claim 1 or claim 2, wherein the plurality of hydrogel particles comprises: a) crosslinked monomers of 2-hydroxyterephthalic acid, tetraethyl dimethacrylate, and acrylic acid, or b) crosslinked monomers of stearyl acrylate, polyethylene glycol) methacrylate, and poly(ethylene gylcol) dimethacrylate.

4. The therapeutic composition of claim 3, wherein the one or more antimicrobial agents is selected from the group consisting of gentamicin, tobramycin, amikacin, neomycin, plazomicin, paromomycin, streptomycin, nitrofurantoin, trimethoprim / sulfamethoxazole, floxacins, cephalosporins, penicillins, or any pharmaceutical salts or combinations thereof.

5. The therapeutic composition of claim 3, wherein the one or more antimicrobial agents is gentamicin.

6. The therapeutic composition of claim 3, wherein the one or more steroid hormones is estradiol.

7. The therapeutic composition of claim 3, wherein the one or more therapeutic agents comprises a combination of gentamicin and estradiol.Attorney ref 340541: 12-25 WO8. The therapeutic composition of claim 3, wherein the one or more antimicrobial agents comprises an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, an anti-parasitic agent, or combinations thereof.

9. The therapeutic composition of any of the preceding claims, wherein the nanogel is able to absorb at least 50% w / w of water.

10. The therapeutic composition of any of the preceding claims, wherein the plurality of hydrogel particles have an average diameter of 20 nm - 200 nm.

11. The therapeutic composition of any of the preceding claims, wherein the plurality of hydrogel particles have an average diameter of 50 nm - 150 nm.

12. The therapeutic composition of any of the preceding claims, wherein the one or more CPPs comprise at least 5 amino acids and equal to or less than 10 amino acids.

13. A method for treating a urinary tract infection (UTI) in a patient comprising administering an effective amount of a therapeutic composition to a patient in need thereof, wherein the therapeutic composition comprises:a) a nanogel comprising a plurality of hydrogel particles having an average diameter from 1 nm to 1,000 nm, wherein each of the hydrogel particles comprise one or more crosslinked polymers;b) one or more cell-penetrating peptides (CPPs) conjugated to the hydrogel particles of the nanogel; andc) one or more therapeutic agents, wherein the one or more therapeutic agents comprises one or more antimicrobial agents, one or more steroid hormones, or combinations thereof, and wherein the one or more therapeutic agents are at least partially encapsulated by the nanogel,wherein administering the effective amount of the therapeutic composition reduces or eliminates bacteria causing the UTI.

14. The method of claim 13, wherein the effective amount of the therapeutic composition is administered directly to a bladder of the patient via a catheter.

15. The method of claim 13 or 14, wherein the plurality of hydrogel particles comprises: a) crosslinked monomers of 2-hydroxyterephthalic acid, tetraethyl dimethacrylate, and acrylicAttorney ref 340541: 12-25 WOacid, or b) crosslinked monomers of stearyl acrylate, polyethylene glycol) methacrylate, and polyethylene gylcol) dimethacrylate.

16. The method of claim 13 or 14, wherein the one or more CPPs comprise a peptide having the sequence CGKRK (SEQ ID NO: 1).

17. The method of claim 16, wherein the one or more antimicrobial agents is selected from the group consisting of gentamicin, tobramycin, amikacin, neomycin, plazomicin, paromomycin, streptomycin, nitrofurantoin, trimethoprim / sulfamethoxazole, floxacins, cephalosporins, penicillins, or any pharmaceutical salts or combinations thereof.

18. The method of claim 17, wherein the one or more antimicrobial agents is gentamicin.

19. The method of claim 18, wherein the effective amount of the therapeutic composition comprises 1 mg / mL of gentamicin.

20. The method of claim 16, wherein the one or more steroid hormones is estradiol.

21. The method of claim 16, wherein the one or more therapeutic agents comprises a combination of gentamicin and estradiol.

22. The method of any of claims 13-21, wherein the administration of the effective amount of the therapeutic composition reduces an amount of intracellular bacterial communities (IBCs) in the patient by at least 80%.

23. The method of any of claims 13-22, wherein the administration of the effective amount of the therapeutic composition reduces an amount of IBCs in the patient by at least 95%.

24. The method of any of claims 13-23, wherein the IBCs in the patient comprise Pseudomonas aeruginosa or Escherichia coli.

25. The method of any of claims 13-24 further comprising administering an initial treatment to the patient and administering the effective amount of the therapeutic composition to the patient at least 48 hours after the initial treatment was ineffective in reducing or eliminating the bacteria causing the UTI.

Images