Peptide tethered particles and methods of use

Abstract

Provided herein are compositions including a functionalized particle, optionally a liposome and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof. Also provided herein are methods of using the functionalized particles in increasing residence time of a therapeutic agent in a target treatment area for use in drug delivery and / or treating, eradicating, or inhibiting biofilm formation.

Description

PEPTIDE TETHERED PARTICLES AND METHODS OF USE CROSS-REFERENCE TORELATEDAPPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 550,339, filed February 06, 2024, and U.S. Provisional Application Serial No. 63 / 643,011, filed May 06, 2024, the entire contents of which are incorporated herein by reference. SEQUENCE LISTING

[0002] A computer-readable form (CRF) sequence listing having file name Sequence_Listing_10738_1120.xml (16,608 Bytes), created on February 2, 2024, is incorporated herein by reference. The nucleic acid sequences listed in the accompanying sequence listing are shown using standard abbreviations as defined in 37 C.F.R. § 1.822. TECHNICAL FIELD

[0003] The present disclosure relates generally to the field of medical therapeutics, specifically compositions and methods for drug delivery and targeting biofilms. BACKGROUND

[0004] Chronic diseases and biofilms are a leading cause of morbidity and mortality worldwide. According to the Centers for Disease Control and Prevention (CDC), six in ten adults in the United States have a chronic disease, and four in ten have two or more. Conditions such as diabetes and chronic inflammatory diseases require long-term medical management, often involving frequent drug administration.

[0005] Diabetic retinopathy, for example, is characterized by pathological neovascularization in the retina and necessitates frequent intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents. However, these repeated injections pose significant risks, including ocular complications and a substantial burden on patients and caregivers. The need for frequent administration stems from the rapid clearance of drugs from the vitreous humor, reducing their bioavailability and therapeutic efficacy. Similarly, in rheumatoid arthritis, intra- articular drug delivery faces limitations due to rapid clearance of therapeutics from the synovial fluid, leading to short residence times ranging from 1 to 25 hours, depending on the drugproperties. The fast drainage of drugs into the lymphatic circulation not only lowers bioavailability but also increases the risk of systemic side effects. While numerous drug delivery systems have been explored to enhance drug retention, many have failed to achieve significant improvements without causing adverse effects, such as inflammation.

[0006] Further, biofilms, or immobilized microbial colonies, cause infections and serious diseases, leading to one-third of global mortality. Roughly more than 60% of hospital-associated infections are attributed to biofilms. Although various antibiotics have been developed, overuse or misuse of antibiotics in the clinics has led to the emergence and spread of antibiotic-resistant bacteria. Infection by antibiotic-resistant bacteria is expected to become the number one cause of death by the year 2050.

[0007] Accordingly, a need exists to identify compositions and treatments for extending drug delivery and / or disrupting biofilm formation. SUMMARY

[0008] The present disclosure generally relates to colloid particles, functionalized with a peptide and methods of using the functionalized particles for extending drug delivery and / or disrupting biofilm formation.

[0009] In one embodiment disclosed herein, the present disclosure is directed to a functionalized particle comprising a liposome; a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof.

[0010] In an embodiment disclosed herein, the present disclosure is directed to a method of treating, eradicating, or inhibiting a biofilm, comprising contacting the biofilm with a functionalized particle comprising a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof.

[0011] In an embodiment disclosed herein, the present disclosure is directed to a method of increasing residence time of a therapeutic agent in a target treatment area in a subject in need thereof, the method comprising administering to the target treatment area of the subject afunctionalized particle comprising: a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof; wherein the liposome at least partially encapsulates the therapeutic agent.

[0012] These and other features, aspects, and advantages will become better understood with reference to the following description and the appended claims.

[0013] Additional features and advantages of the embodiments described herein will be set forth in the detailed description that follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description that follows, the claims, as well as the appended drawings. BRIEFDESCRIPTION OF THEDRAWINGS

[0014] The objects and advantages of the present disclosure will be further appreciated in light of the accompanying drawings, in which:

[0015] FIG. 1A-FIG. 1B demonstrate the effect of HABP-functionalized liposomes on biofilm growth (FIG. 1A) and biofilm metabolism (FIG. 1B).

[0016] FIG. 2 illustrates the biofilm eradication efficacy of HABP liposomes, showing nearly complete biofilm removal (~0% residual biofilm) at a concentration of 3 mM.

[0017] FIG. 3A-FIG. 3B graphically depicts dye diffusion through hyaluronic acid using free dye, dye encapsulated in bare liposomes, and dye in HABP liposomes, in vitro (left panel) and ex vivo (right panel).

[0018] FIG. 4A-FIG. 4D graphically depict dose-dependent cytotoxicity results of Dex- loaded bare liposome (FIG. 4A) and Dex-loaded HABP liposome on TM cells as determined by MTT assays (FIG.4B), Dex-loaded bare liposome (FIG. 4C) and Dex-loaded HABP liposome on TM cells as determined by LDH assays (FIG. 4D).

[0019] FIG. 5 graphically depicts the IL-6 concentration of the supernatant of human retinal microvascular endothelial cells using various concentrations of dex alone, dex-loaded bare liposomes, and dex-loaded HABP liposomes.DETAILEDDESCRIPTION

[0020] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document.

[0021] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently- disclosed subject matter belongs.

[0023] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0024] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0025] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0026] The term “administering,” as used herein, refers to any route of administering an effective amount of a therapeutic agent. In embodiments, the administering includes, but is not limited to, intraocularly, intravitreally, intravenous, intraperitoneal, subcutaneous, intramuscular, oral, intracerebral, intraspinal, intrathecal, subarachnoid, epidural, periocular, intraocular administration, intra-articular, and the like. In a specific embodiment, the therapeutic agent is administered intraocularly, intravitreally, or intra-articular.

[0027] The terms “treat,” “treatment,” and “treating,” as used herein, refer to a method of alleviating or abrogating a disease, disorder, and / or symptoms thereof in a subject, including a mammal. In certain embodiments, the subject is a human patient.

[0028] The present disclosure generally relates to colloid particles that bind to a surrounding biological environment. Generally, the particles of the instant disclosure are tethered to one or more ligands. The tethered particles may be used to enhance drug delivery and / or inhibit biofilm formation. As will be demonstrated herein, the particles of the present disclosure, such as liposomes conjugated with hyaluronic acid binding peptides (HABP) via a 3.4k PEG linker reduced biofilm formation by 27% without the use of antibiotics. Other particles, such as liposomes functionalized with HABP through a 5k PEG linker exhibited prolonged residence time, remaining localized for several days, in contrast to their non-functionalized counterparts, which dissipated within hours.

[0029] As used herein, a “colloid particle” refers to a particle that is dispersed within a medium, but does not settle out over time due to its size and / or interaction with the dispersing medium. Optionally, the particles range in size from approximately 1-1000 nm, including about 100 nm, about 200 nm, about 250 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 750 nm, about 800 nm, about 900 nm, about 1000 nm, and any subrange having endpoints defined by any two of the aforementioned values.

[0030] Exemplary, non-limiting examples of colloid particles include liposomes, ethosomes, micelles, niosomes, nanoemulsions, solid lipid nanoparticles, polymeric nanoparticles, dendrimers, combinations thereof, and the like, though any suitable particle is contemplated and possible. Optionally, the colloidal particle is a liposome.

[0031] The colloidal particles may be functionalized to modulate various properties, including drug-delivery, tissue engineering, and regenerative medicine. In some embodiments, apeptide is tethered to the colloidal particle. The peptide may be tethered to the particle by any suitable means, including, but not limited to covalent grafting, self-assembly on the particles, adsorption, conjugation, etc. In some embodiments, the peptide is conjugated to the colloid particle via a polyethylene glycol (PEG) linker. Optionally, the PEG linker has a molecular weight of about 1k to about 10k, including about 1k, about 2k, about 2.5k, about 3k, about 3.5k, about 4k, about 4.5k, about 5k, about 5.5k, about 6k, about 6.5k, about 7k, about 7.5k, about 8k, about 8.5k, about 9k, about 9.5k, and about 10k, including any subrange having endpoints defined by any two of the aforementioned values. Optionally, the PEG linker is functionalized with a maleimide functional group.

[0032] Without being bound by theory, tethering one or more peptides to the particle may help target specific environments, improve cellular uptake, enhance biocompatibility, improve cellular adhesion, and / or control drug release. Optionally, the peptide is a hyaluronic acid binding protein, a triple helix forming protein, or a combination thereof.

[0033] In some embodiments, the peptide tethered to the colloidal particle is a hyaluronic acid binding protein. Hyaluronic acid binding proteins (HABPs) are proteins that interact with hyaluronic acid (HA), a major component of the extracellular matrix. Hyaluronic acid binding protein (HABP) as used herein refers to peptides that have a linear 9-15 residue HA-binding motif containing multiple basic amino acids and bind to HA via this motif. This HA-binding motif (SEQ ID NO:1) may have a B-X7-B motif, wherein B is any basic amino acid (e.g., arginine, lysine, or histidine) and X is a non-acidic amino acid, that is any amino acid excluding aspartate and glutamate. In some embodiments, the HABP is the binding motif of link protein, CD44, aggrecan, versican, LYVE-1, TSG-6, RHAMM, IHABP, cdc37, P-32, SPACR, and / or SPACRCAN. In some embodiments, the HABP has the amino acid sequence STMMSRSHKTRSHHV (SEQ ID NO:2), TMTRPHFHKRQLVLS (SEQ ID NO:3), and / or RRDDGAHWQFNALTVR (SEQ ID NO:4), or a peptide having 85% identity therewith, wherein the binding motif is conserved. In some embodiments, the HABP has the amino acid sequence STMMSRSHKTRSHHV (SEQ ID NO:2).

[0034] In some embodiments, the peptide tethered to the colloidal particle is a triple helix forming protein. Triple helix forming proteins (THFP) are proteins that mimic collagen’s triple-helical structure and are capable of binding to denatured collagen strands. As used herein, THFPs refer to peptides having a general binding sequence (GPO)n (SEQ ID NO:5), where n is an integer from 6–10, G is glycine, P is proline, and O is hydroxyproline.

[0035] In some embodiments, the functionalized particle encapsulates or partially encapsulates a therapeutic agent. As used herein, a “therapeutic agent” refers to any compound, molecule, composition, biologic, or formulation that is capable of treating, alleviating, preventing, or diagnosing a disease, disorder, or medical condition in a subject. Therapeutic agents may include, but are not limited to, small molecules, peptides, proteins, antibodies, nucleic acids, gene therapies, vaccines, biologics, cell-based therapies, and any pharmaceutically acceptable derivatives, salts, or formulations thereof. In some embodiments, the therapeutic agent is an approved or experimental treatment for one or more conditions. Exemplary, non-limiting conditions include diabetic retinopathy, wet age-related macular degeneration, retinal vein occlusion, diabetic macular edema, abnormal blood vessel growth in the eye from extreme near- sightedness, ocular histoplasmosis, retinal detachment, rheumatoid arthritis, degenerative cartilage, synovitis, osteoarthritis, bursitis, gouty arthritis, frozen shoulder syndrome, tendinitis, gout, psoriatic arthritis, axial spondyloarthritis, and juvenile arthritis.

[0036] In some embodiments, the functionalized particles increase the residence time of a therapeutic agent in a target treatment area of a subject. It will be appreciated that the peptide tethered to the colloid particle may be adjusted based on the molecular environment of the target treatment area. For example, these compositions may be designed to bind either to denatured collagen in the joint space or to HA in the vitreous humor, preventing rapid clearance.

[0037] For example, and without being bound by theory, THFP-tethered colloid particles may provide a robust anchoring mechanism for prolonging drug retention in tissues affected by chronic diseases. Optionally, the target treatment area comprises denatured collagen. As shown herein, the present disclosure demonstrates the use of triple-helix forming peptides (THFP) to enhance the binding affinity of the colloid particles to collagen-rich tissues. Triple-helix interactions exhibit dissociation constants in the nanomolar range (e.g., 30 nM for type VI collagen), which is approximately 1000 times lower than typical protein-protein interactions.

[0038] Further, and without being bound by theory, HABP functionalized particles may enhance drug residence time. For example, hyaluronic acid is abundant in synovial fluid of joints,the vitreous body, and other connective tissues, making it a viable target for prolonged drug retention. Optionally, the target treatment area is the vitreous of an eye or a joint space. HABPs recognize HA through the conserved binding motif (B(X7)B, mimicking native HA-protein interactions, and enabling the particle to remain in the targeted location for a longer period of time.

[0039] In some embodiments, the therapeutic agent is controllably released from the functionalized particle by any suitable mechanism (e.g., ultrasound, light / laser activation, heat etc.). In some embodiments, the mechanism comprises near-infrared radiation. For example, and without being bound by theory, the colloid particle may have nano valves integrated into the particles that reversibly open and close in response to a stimulus, inducing a conformational change that allows release of the encapsulated therapeutic agent. In some embodiments, the stimulus may trigger an irreversible structural breakdown of the particle, leading to a burst release of the therapeutic agent. In other embodiments, the therapeutic agent may not be triggered to burst release and may instead slowly release from the colloid particle, e.g., via diffusion.

[0040] In some embodiments, the functionalized particles treat, inhibit, or eradicate the formation of biofilms. In some embodiments, the functionalized particle disrupts bacterial adhesion via blocking cellular pathways and physically hindering bacterial interaction. For example, and without being bound by theory, upon binding to the extracellular polymeric substances (EPS) in a biofilm matrix, the colloid particles may migrate deeper into the biofilm matrix, a process primarily driven by diffusion, thereby sterically preventing signaling between the cells. While particle accumulation within biofilms results from both initial attachment and subsequent migration, these processes may occur simultaneously rather than sequentially. Extracellular polymeric substances (EPS) include, but are not limited to, polysaccharides (such as exopolysaccharides), proteins, lipids, and extracellular DNA. The components of EPS are of microbial origin and are involved in interactions between the bacterial cells of a biofilm and their environment.

[0041] The compositions provided herein may be used to block EPS signaling pathways, disrupt focal adhesion, and sterically hinder the biofilm. In some embodiments, the tethered particles continuously deposit onto the outer biofilm surface as others penetrate deeper into the matrix, creating a dynamic equilibrium. Unlike electrostatic interactions observed with positively charged nanoparticles, the data provided herein isolates the physical impact of the particle presence. The experimental results demonstrate that HABP-functionalized liposomes significantlyinhibit biofilm growth and metabolism, with a minimal biofilm inhibition concentration (MBIC) of 0.05 mM—100 times lower than that of bare liposomes. HABP liposomes also exhibit stronger biofilm eradication, with a minimum biofilm eradication concentration (MBEC) of <0.05 mM, compared to 7 mM for bare liposomes. The findings highlight the importance of steric hindrance in preventing biofilm formation and eradicating established biofilms, offering a promising antimicrobial-free strategy to combat biofilm-associated infections. EXAMPLES

[0042] The following Examples are offered by way of illustration and are presented in a manner such that one skilled in the art should recognize are not meant to be limiting to the present disclosure as a whole or to the appended claims.

[0043] Example 1: Preparation and characterization of functionalized liposomes

[0044] To form the functionalized liposomes, a peptide having SEQ ID NO: 2 was conjugated to a maleimide-functionalized polyethylene glycol-lipid (MAL-PEG-lipid) via a thiol- maleimide reaction, ensuring site-specific tethering through a cysteine residue present in the peptide sequence. The resulting peptide-MAL-PEG-lipid conjugate was subsequently incorporated into the lipid bilayer during the formation of liposomal nanoparticles. The particles were characterized, the details of which are described in Table 1, including the size, polydispersity index, zeta potential and %EE.

[0045] Table 1: Summary of Size, PDI, Zeta potential and EE%

[0046] Example 2: Inhibition and Eradication of Biofilm by HABP-Functionalized Liposomes

[0047] To evaluate biofilm inhibition, bacterial cultures were incubated with varying concentrations of HABP-functionalized liposomes, bare liposomes, or free HABP. The extent of biofilm formation was quantified using two complementary assays: the crystal violet (CV) assay, which measures total biofilm biomass, and the triphenyl tetrazolium chloride (TTC) assay, which assesses metabolic activity within biofilms.

[0048] FIGs. 1A-1B demonstrate that liposomes functionalized with hyaluronan-binding peptide (HABP) effectively inhibited bacterial biofilm growth and metabolism, reducing biofilm formation to approximately 10–20% of the control. This inhibition was more pronounced than that observed with either non-functionalized (bare) liposomes or HABP alone, indicating a synergistic effect of peptide functionalization on liposomal activity. The CV assay (FIG. 1A) revealed that HABP-functionalized liposomes exhibited a minimum biofilm inhibition concentration (MBIC) of 0.05 mM, a significantly lower threshold compared to bare liposomes, which had an MBIC of 1.07 mM. Similarly, in the TTC assay (FIG. 1B), HABP-functionalized liposomes reduced biofilm metabolic activity by nearly 40% relative to bare liposomes, further supporting their superior biofilm inhibitory properties.

[0049] As demonstrated, HABP-functionalized liposomes exhibited strong affinity for EPS, leading to prolonged retention within the biofilm microenvironment and enhanced sterichindrance effects. By contrast, bare liposomes, lacking specific EPS-binding capabilities, exhibited reduced localization within the biofilm matrix and correspondingly lower inhibitory efficacy.

[0050] The density of functionalized liposomes within the biofilm matrix also contributed to inhibition efficacy, as increased concentrations result in greater steric interference with bacterial adhesion and surface colonization. Interestingly, the CV assay indicated that free HABP alone had only a modest effect on biofilm formation (~80% biofilm growth compared to control), whereas the TTC assay revealed that free HABP significantly suppressed biofilm metabolic activity to below 50% at a concentration of 0.01 mM (FIG. 1B). This discrepancy may arise from the nonspecific nature of crystal violet staining, which detects not only intact biofilms but also associated biomolecules such as extracellular DNA, dead cells, and protein debris. Notably, the ability of HABP to inhibit biofilm formation without relying on conventional antimicrobial agents is unexpected, as it may be used in treatments where there is a concern related to antimicrobial resistance. The superior biofilm suppression observed with HABP-functionalized liposomes, compared to free HABP, further highlights the role of steric hindrance in biofilm disruption.

[0051] To assess biofilm eradication and the minimum biofilm eradication concentration (MBEC), mature biofilms were allowed to develop under static conditions before treatment with increasing concentrations of either HABP-functionalized liposomes, bare liposomes, or free HABP, as shown in FIG. 2.

[0052] Biofilm removal was quantified using CV staining to evaluate biomass reduction and TTC staining to assess metabolic activity post-treatment. FIG. 2 depicts the effect of HABP- functionalized liposome concentration on percent biofilm metabolism, demonstrating nearly complete biofilm removal (~0% residual biofilm) at a concentration of 3 mM. This eradication efficiency was approximately 30% greater than that of bare liposomes, underscoring the significant contribution of HABP functionalization.

[0053] The minimum biofilm eradication concentration (MBEC) was determined to be <0.05 mM for HABP-functionalized liposomes, in contrast to the significantly higher MBEC of 7 mM required for bare liposomes. Additionally, free HABP alone exhibited substantial biofilm metabolic reduction (~20–40%), but its incorporation into liposomes further enhanced this effect, emphasizing the critical role of steric hindrance in biofilm disruption.

[0054] Collectively, these findings highlight the efficacy of HABP-functionalized liposomes as a potent biofilm-inhibitory and biofilm-eradicating platform. The dual mechanism of action—EPS targeting and steric hindrance—distinguishes this approach from conventional antimicrobial strategies.

[0055] Example 3: In vitro and ex-vivo diffusion study of free Cy5, Cy5-loaded bare and HABP-liposome

[0056] To assess the release kinetics and retention of dexamethasone (Dex)-loaded bare and hyaluronic acid-binding peptide (HABP)-functionalized liposomes, an elution study was conducted using a 0.65 μm Amicon filtration device. The study was designed to simulate daily drug release, with an initial Dex concentration of ~140 μM (72.24 μg / mL), monitoring elution over a 14-day period under conditions mimicking physiological drug diffusion (10 μM Dex, ~5.16 μg / mL per day).

[0057] Preparation of HA Solution and Filtration Setup

[0058] A total of 0.7 mL of hyaluronic acid (HA, Mw ~1–2 MDa) solution was prepared in an Eppendorf tube. An additional 300 μL of HA solution was introduced into an Amicon filtration device (pore size 0.65 μm), which was subsequently inserted into the Eppendorf tube containing the initial HA solution.

[0059] Liposomal Drug Loading and Incubation

[0060] Dex-loaded bare and HABP-functionalized liposomes (~140 μM Dex, 72.24 μg) were added to the Amicon filtration device, allowing interaction with the HA matrix.

[0061] Collection and Centrifugation

[0062] Following elution, the HA solution containing Dex-loaded liposomes was collected and subjected to ultracentrifugation at ~100,000 rpm for 1 hour to sediment the liposomes.

[0063] Quantification of Dex Retention and Release

[0064] The sedimented Dex-loaded bare and HABP-functionalized liposomes were resuspended in 0.2 mL HEPES buffer. The absorbance of Dex was measured to determineretention efficiency. Daily elution of Dex from the liposomes was recorded over a 14-day period, replacing the HA-containing solution in the Eppendorf tube daily. A cumulative release profile of Dex-loaded bare and HABP-functionalized liposomes was generated.

[0065] Diffusion Study

[0066] Parallel diffusion studies were performed in a 35 mm dish containing 5 mL HA solution (4 mg / mL, Mw 1–2 MDa) dissolved in PBS buffer and in an ex vivo rabbit eye. Cy5- labeled liposomes were used to visualize diffusion behavior. Specifically, 10 μL (0.6293 μg of Cy5) of Cy5, Dex / Cy5-bare-liposomes, and Dex / Cy5-HABP-liposomes (Cy5 concentration: 62.93 μg / mL) were injected into the HA solution.

[0067] Diffusion was monitored at multiple time points (0 min, 10 min, 20 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h) using Micron IV in vivo microscopy, and fluorescence intensity was quantified using ImageJ software. These results are shown in FIG. 3 for both the in vitro (left panel) and ex vivo (right panel) diffusion studies.

[0068] The findings highlight the potential of HABP-functionalized liposomes as an advanced strategy for enhancing drug retention in chronic disease treatment. By exploiting the high-affinity interactions of triple-helix structures and HA-binding motifs, this approach offers a means to improve drug localization and prolong therapeutic effects. Additionally, the incorporation of a light-triggered release mechanism provides precise control over drug kinetics. This strategy holds significant promise for improving the efficacy and patient adherence of treatments for conditions such as diabetic retinopathy and rheumatoid arthritis, thereby reducing the burden of frequent injections and associated complications.

[0069] Example 4: Cytotoxicity Assessment Using MTT and Lactate Dehydrogenase (LDH) Assays

[0070] The cytotoxic effects of dexamethasone (Dex)-loaded bare liposomes and Dex- loaded HABP-functionalized liposomes were evaluated using the MTT and LDH assays.

[0071] Cell Culture and Treatment

[0072] Trabecular meshwork (TM) cells were seeded in 96-well plates at a density of 1×104cells per well and incubated overnight to allow for cell attachment. The cells were thentreated with Dex-loaded bare liposomes or Dex-loaded HABP-functionalized liposomes at Dex concentrations of 10 nM, 100 nM, 1 µM, 10 µM, and 20 µM. The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. Every 48 hours, the culture medium was replaced with fresh medium containing the respective Dex-loaded liposomes. After six days of treatment, the cytotoxic effects were assessed.

[0073] LDH Assay

[0074] Following the treatment period, the culture medium was collected from each well for the LDH assay. Additional wells were included as controls for spontaneous LDH activity (treated with 10 μL of sterile ultrapure water) and maximum LDH activity (treated with 10 μL of 10× lysis buffer). The collected samples were processed according to the manufacturer's instructions to determine LDH release as an indicator of cell membrane integrity and cytotoxicity.

[0075] MTT Assay

[0076] After collecting the culture medium for the LDH assay, fresh medium containing MTT reagent (5 mg / mL) was added to each well and incubated for an additional 4 hours at 37°C in 5% CO2. The medium was then removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the intracellular formazan crystals. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Power Wave XS; BioTek, Winooski, VT).

[0077] Data Analysis

[0078] Cell viability (%) was calculated based on the absorbance values obtained from the MTT assay using the following equation: % Cytotoxicity = (compound treated LDH activity – spontaneous LDH activity)÷ (maximum LDH activity – spontaneous LDH activity)*100.

[0079] Each experimental condition was performed in quadruplicate (n=4), and the means and standard deviations (SDs) were determined. The results were analyzed to compare the cytotoxic effects of Dex-loaded bare and HABP-functionalized liposomes at different concentrations. The results are shown in FIGs. 4A-4D.

[0080] These results demonstrate the safety of the functionalized particles to non-targeted cells.

[0081] Example 5: In Vitro Anti-Inflammatory Activity

[0082] TNF-α–Induced Inflammation in HRMECs: Analysis of Proinflammatory Markers Using ELISA

[0083] The anti-inflammatory effects of free dexamethasone (dex), dex-loaded bare liposomes, and dex-loaded HABP-functionalized liposomes were evaluated using primary human retinal microvascular endothelial cells (HRMECs) (ACBRI 181; Cell Systems Corporation, Kirkland, WA, USA). The study was conducted with slight modifications to a previously published protocol.

[0084] Cell Culture and Experimental Setup

[0085] HRMECs were cultured in Endothelial Cell Growth Medium (C-22010; Promocell, Heidelberg, Germany), supplemented with all necessary growth factors and antibiotics (Cell Systems). Cells were maintained in 60 mm culture dishes at 37°C in a humidified atmosphere with 5% CO2. Upon reaching 70–80% confluence, the cells were trypsinized, counted, and seeded into 24-well plates at a density of 1.72×104 cells per well.

[0086] Once the cells reached 70–80% confluence in the 24-well plates, they were exposed to an inflammatory stimulus by treatment with 10 ng / mL recombinant human TNF-α (H8916, Sigma Aldrich) in a starvation medium consisting of endothelial basal medium supplemented with 0.5% fetal bovine serum (FBS). The treatment was performed for 24 hours in the presence or absence of free Dex, Dex-loaded bare liposomes, or Dex-loaded HABP- functionalized liposomes at Dex concentrations of 10 nM, 100 nM, and 10 µM.

[0087] Measurement of IL-6 Levels Using ELISA

[0088] Following the treatment period, the culture supernatant was collected from each well, and the levels of the proinflammatory cytokine interleukin-6 (IL-6) were quantified using IL-6 ELISA kits, following the manufacturer’s instructions. These results are depicted in FIG. 5.

[0089] As shown, the HABP functionalized liposomes lowered the IL-6 levels more than the bare liposomes or dexamethasone alone, at all concentrations.

[0090] In autoimmune diseases (e.g., rheumatoid arthritis, lupus), high IL-6 drives persistent inflammation. IL-6 plays a role in triggering acute-phase responses (e.g., fever, C- reactive protein (CRP) production). In severe infections (e.g., COVID-19, sepsis), high IL-6 contributes to cytokine storm, leading to tissue damage and organ failure. Thus, these results indicate that the functionalized particles may treat or improve various diseases associated with inflammation.

[0091] Aspect Listing

[0092] In a first aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle comprising a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof.

[0093] In a second aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein the liposome at least partially encapsulates a therapeutic agent.

[0094] In a third aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein release of the therapeutic agent from the liposome is triggered by light.

[0095] In a fourth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle wherein the THFP comprises a binding sequence (GPO)n, wherein: n = an integer from 6-10; G is glycine; P is proline; and O is hydroxyproline.

[0096] In a fifth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein the HABP comprises a binding sequence B(X7)B, wherein B is histidine (H), arginine (R) or lysine (K); and X is any non-acidic amino acid.

[0097] In a sixth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein the peptide has an amino acid sequence according to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0098] In a seventh aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein the peptide has an amino acid sequence according to SEQ ID NO:2.

[0099] In an eighth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a functionalized particle, wherein the peptide is tethered to the liposome by a polyethylene glycol linker.

[0100] In a ninth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method of treating, eradicating, or inhibiting a biofilm, comprising contacting the biofilm with the functionalized particle of any other aspect herein.

[0101] In a tenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the biofilm comprises an extracellular polymeric substance (EPS).

[0102] In an eleventh aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the EPS comprises one or more components selected from polysaccharides, proteins, lipids, or extracellular DNA.

[0103] In a twelfth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the functionalized particle binds to the one or more components of the EPS, thereby sterically hindering bacterial cell signaling within the biofilm.

[0104] In a thirteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method of increasing residence time of a therapeutic agent in a target treatment area in a subject in need thereof, the method comprising administering to the target treatment area of the subject a functionalized particle comprising a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof; wherein the liposome at least partially encapsulates the therapeutic agent.

[0105] In a fourteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the target treatment area is a vitreous of an eye or a joint space.

[0106] In a fifteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the target treatment area comprises denatured collagen.

[0107] In a sixteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the subject is suffering from a condition selected from the group consisting of diabetic retinopathy, wet age-related macular degeneration, retinal vein occlusion, diabetic macular edema, abnormal blood vessel growth in the eye from extreme near-sightedness, ocular histoplasmosis, retinal detachment, rheumatoid arthritis, degenerative cartilage, synovitis, osteoarthritis, bursitis, gouty arthritis, frozen shoulder syndrome, tendinitis, gout, psoriatic arthritis, axial spondyloarthritis, and juvenile arthritis.

[0108] In a seventeenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, further comprising applying a stimulus to the target treatment area to trigger release of the therapeutic agent from the functionalized particle.

[0109] In an eighteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein applying the stimulus comprises exposing the treatment area to a light source.

[0110] In a nineteenth aspect, alone or in combination with any other aspect herein, the present disclosure concerns a method, wherein the stimulus is near-infrared radiation.

[0111] It is noted that the terms “substantially” and “about” may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. The term “substantially” is used herein also to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. Thus, it is used to represent the inherent degree of uncertainty that may be attributed to any quantitativecomparison, value, measurement, or other representation, referring to an arrangement of elements or features that, while in theory would be expected to exhibit exact correspondence or behavior, may in practice embody something less than exact.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting. As used in the specification and appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0113] It is noted that one or more of the following claims utilize the term “wherein” as a transitional phrase. For the purposes of defining the present technology, it is noted that this term is introduced in the claims as an open-ended transitional phrase that is used to introduce a recitation of a series of characteristics of the structure and should be interpreted in like manner as the more commonly used open-ended preamble term “comprising.”

[0114] It should be understood that where a first component is described as “comprising” or “including” a second component, it is contemplated that, in some embodiments, the first component “consists” or “consists essentially of” the second component. Additionally, the term “consisting essentially of” is used in this disclosure to refer to quantitative values that do not materially affect the basic and novel characteristic(s) of the disclosure.

[0115] It should be understood that any two quantitative values assigned to a property or measurement may constitute a range of that property or measurement, and all combinations of ranges formed from all stated quantitative values of a given property or measurement are contemplated in this disclosure.

[0116] While particular embodiments have been illustrated and described herein, it should be understood that various other changes and modifications may be made without departing from scope of the claimed subject matter. Moreover, although various aspects of the claimed subject matter have been described herein, such aspects need not be utilized in combination. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the claimed subject matter.

Claims

CLAIMS1. A functionalized particle comprising: a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple- helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof.

2. The functionalized particle of claim 1, wherein the liposome at least partially encapsulates a therapeutic agent.

3. The functionalized particle of claim 2, wherein release of the therapeutic agent from the liposome is triggered by light.

4. The functionalized particle of claim 1, wherein the THFP comprises a binding sequence (GPO)n, wherein: n = an integer from 6-10; G is glycine; P is proline; and O is hydroxyproline.

5. The functionalized particle of claim 1, wherein the HABP comprises a binding sequence B(X7)B, wherein B is histidine (H), arginine (R) or lysine (K); and X is any non-acidic amino acid.

6. The functionalized particle of claim 1, wherein the peptide has an amino acid sequence according to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

7. The functionalized particle of claim 1, wherein the peptide has an amino acid sequence according to SEQ ID NO:

2.

8. The functionalized particle of claim 1, wherein the peptide is tethered to the liposome by a polyethylene glycol linker.

9. A method of treating, eradicating, or inhibiting a biofilm, comprising contacting the biofilm with the functionalized particle of claim 1.

10. The method of claim 9, wherein the biofilm comprises an extracellular polymeric substance (EPS).

11. The method of claim 10, wherein the EPS comprises one or more components selected from polysaccharides, proteins, lipids, or extracellular DNA.

12. The method of claim 11, wherein the functionalized particle binds to the one or more components of the EPS, thereby sterically hindering bacterial cell signaling within the biofilm.

13. A method of increasing residence time of a therapeutic agent in a target treatment area in a subject in need thereof, the method comprising administering to the target treatment area of the subject a functionalized particle comprising: a liposome; and a peptide tethered to the liposome, wherein the peptide is selected from a triple-helix forming peptide (THFP), a hyaluronic acid binding peptide (HBAP), or a combination thereof; wherein the liposome at least partially encapsulates the therapeutic agent.

14. The method according to claim 13, wherein the target treatment area is a vitreous of an eye or a joint space.

15. The method according to claim 13, wherein the target treatment area comprises denatured collagen.

16. The method according to claim 13, wherein the subject is suffering from a condition selected from the group consisting of diabetic retinopathy, wet age-related macular degeneration, retinal vein occlusion, diabetic macular edema, abnormal blood vessel growth in the eye from extreme near-sightedness, ocular histoplasmosis, retinal detachment, rheumatoid arthritis, degenerative cartilage, synovitis, osteoarthritis, bursitis, gouty arthritis, frozen shoulder syndrome, tendinitis, gout, psoriatic arthritis, axial spondyloarthritis, and juvenile arthritis.

17. The method according to any of claims 12-16, further comprising applying a stimulus to the target treatment area to trigger release of the therapeutic agent from the functionalized particle.

18. The method according to claim 17, wherein applying the stimulus comprises exposing the treatment area to a light source.

19. The method according to claim 17, wherein the stimulus is near-infrared radiation.

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