Elastin-like polypeptides for non-covalent capture of vescalagin and enhanced permeation through nasal mucosa

Abstract

A composition and method of using the composition to treat neurological disorders including Alzheimer's disease are disclosed wherein the composition includes an elastin-like polypeptide (ELP) and a drug. The ELP undergoes a transition from a solution phase to a gel phase as the temperature is raised above a transition temperature, and in doing so sequesters at least a portion of the drug into the gel phase. The composition is configured such that intranasal delivery of the composition results in gelation and sequestration of the drug by the ELP, thereby allowing the drug to traverse olfactory mucosa into brain tissue in order to treat neurological disorders, including Alzheimer's disease.

Description

ELASTIN-LIKE POLYPEPTIDES FOR NON-COVALENT CAPTURE OF VESCALAGIN AND ENHANCED PERMEATION THROUGH NASAL MUCOSACROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 710,786, filed October 23, 2024, the disclosures of which are hereby incorporated by reference in their entireties.TECHNICAL FIELD

[0002] The present disclosure relates to the intranasal delivery of drugs targeting the brain, specifically for the treatment of Alzheimer’s disease.BACKGROUND

[0003] The effectiveness of therapeutics that target Alzheimer’s disease (AD) pathology is often limited to a drug’s inability to migrate through the blood-brain barrier (BBB). The BBB comprises endothelial cells and astrocytes and permits only small molecules to diffuse from blood capillaries to neural tissue. This limits the selection of therapeutics that can be effectively delivered to the brain using oral or IV routes. Nearly all FDA-approved therapeutics for AD are delivered orally or intravenously, which results in systemic exposure of the therapeutic, requires more concentrated doses, and can lead to adverse off-target effects. These delivery routes are also limited by expeditious drug clearance via the kidneys and liver, necessitating more frequent dosages and increased patient compliance. One class of drugs that fail to permeate through the BBB and are susceptible to quick clearance is the class of polyphenolic compounds. Polyphenols interact with A|3 plaques through 7t-7t interactions with diphenylalanine, a peptide involved in A(3 fibril assembly. Polyphenols also act as chelating agents to remove metals like zinc and copper that promote plaque formation and serve as antioxidants, neutralizing ROS and restoring normal autophagic processes in neural tissue affected by Alzheimer's disease. Vescalagin (VSG) is a polyphenolic ellagitannin derived from oak and cork trees shown to reduce ROS mediated cell damage of L929 fibroblast cells, prevent Ap-mediated cell death of SHSY5Y neuroblastomacells, and prevent and reverse the assembly of Ap plaques in vitro. While studies show great promise for VSG acting as an AD therapeutic, this molecule is too large to diffuse through the BBB. Thus, VSG is an example of a promising AD therapeutic that is unlikely to pass through the BBB via oral and IV delivery routes. To address the growing need for potentially efficacious AD therapeutics such as VSG, better delivery mechanisms must be pursued to improve their in vivo efficacy.SUMMARY

[0004] In some embodiments, a composition including an elastin-like peptide (ELP) is disclosed. The ELP undergoes a transition from a solution phase to a gel phase as the temperature is raised above a sol-gel transition temperature. The composition further includes a drug, wherein the ELP sequesters at least a portion of the drug into the gel phase when the temperature is raised above the sol-gel transition temperature. The ELP is configured such that intranasal delivery of the composition results in gelation and sequestration of the drug by the ELP, wherein gelation of the ELP and sequestration of the drug by the ELP upon intranasal delivery of the composition allows the drug to traverse olfactory mucosa into brain tissue.

[0005] According to some embodiments, methods are disclosed for the intranasal delivery of such compositions in order to target the drug to neural tissue for the treatment of neurological disorders, including Alzheimer’s disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figs. 8-10 are submitted as color drawings.

[0007] Fig. 1 shows a cuvette containing ELP-Y4 when heated from below the sol-gel transition temperature Tt to above Tt.

[0008] Fig. 2 shows experimental turbidity curves for ELP-Y4 and ELP-F4.

[0009] Fig. 3 shows experimental turbidity curves for ELP-Y4L4 and ELP-Y4L4-IOXR

[0010] Fig. 4 shows the protective effect of VSG against oxidative damage by H2O2.

[0011] Fig. 5 shows the experimentally determined concentration of VSG for in phosphate buffered saline immediately after adding to VSG to a concentration of 50 pM to 50 pM solutions of ELP-Y4L4 and ELP-Y4L4-IOXR.

[0012] Fig. 6 shows the experimentally determined concentration of VSG remaining in the supernatant for the solution of Fig. 5 following heating to 40 °C and centrifugation.

[0013] Fig. 7 shows the experimentally determined concentration of VSG in the centrifuged pellet for the solution of Fig. 5 following heating to 40 °C and centrifugation.

[0014] Fig. 8 shows confocal fluorescent microscopy of RITC-labeled Y4L4 (red) combined with adhered RPMI 2650 cells, rendered fluorescent (green) due to calcein metabolic processing.

[0015] Fig. 9 shows confocal fluorescent microscopy of RITC-labeled Y4L4-IOXR (red) combined with adhered RPMI 2650 cells, rendered fluorescent (green) due to calcein metabolic processing. Dashed circles show uptake of RITC-labeled Y4L4-IOXR into the RPMI 2650 cells.

[0016] Fig. 10 shows confocal fluorescent microscopy of RITC-labeled Y4L4-IOXR (red) combined with adhered RPMI 2650 cells, rendered fluorescent (green) due to calcein metabolic processing. Dashed circles show how RITC-labeled Y4L4-IOXR self-assembles around the surface of the RPMI cells.

[0017] Fig. 11 demonstrates how RITC-labeled Y4L4 and RITC-labeled Y4L4-IOXR are trapped in the apical transwell compartment and then slowly traverse to the basolateral transwell compartment.DETAILED DESCRIPTION

[0018] Intranasal (IN) drug delivery to the brain is a promising alternative to oral and IV delivery routes since the olfactory mucosa lining the nasal cavity provide a direct access point to neural tissue. Olfactory nerves transcend from the central nervous system’s (CNS) olfactory bulb to the superior epithelial tissue in the nasal cavity where they interact with odorants. This anatomy provides a direct route for therapeutic transport from the nasal mucosa to the brain, devoid of the BBB. Because there is no need to pass the BBB using the IN route, larger and more efficacious therapeutics may be delivered to the brain. However, one of the greatest challenges regarding IN drug delivery is transporting therapeutics through the mucous membrane and preventing mucociliary clearance. Particles trapped in the mucous membrane are transported to the gut. The mucous membrane barrier impedes IN drug delivery since therapeutics displaced to the gut are then delivered systemically. In situ gelling systems provide a means to overcome this limitation since they can solidify following contact with the nasal mucosa, minimizingmucociliary clearance. However, current IN gelling systems rely on complex formulations comprising detergents, lipids, viscosity enhancers, and mucoadhesives. Additionally, these thermo- and pH-responsive gelling systems comprise polymers unnatural to the human body, including poloxamers, chitosan, gellan gum, and pectin.

[0019] One poorly explored class of materials for IN application is the class of elastinlike polypeptides (ELPs). ELPs are recombinant proteins composed of repeat structural motifs derived from tropoelastin, such as the (VPGXG)n pentapeptide sequence (pps), where X can be any amino acid except for proline. This pps is bioinert due to its naturally derived origin and also elicits a natural gelation or liquid-liquid phase separation behavior at a precise transition temperature (Tt). ELPs delivered alone (no drug) via the IN route had a significantly reduced systemic circulation and increased extravascular accumulation in the brain compared to IV delivery of ELPs. However, IN delivery resulted in lung accumulation due to aspiration due to using ELPs that did not phase-separate at nasal passage temperatures (34°C). Additionally, no study has implemented ELP materials to bind therapeutics for IN drug delivery.

[0020] For some embodiments, a composition is disclosed which includes an elastin-like peptide (ELP) and a drug. For such embodiments, the composition is configured such that IN delivery of the composition results in gelation of the composition and sequestration of the drug by the composition. For such embodiments, gelation of the composition and sequestration of the drug upon IN delivery of the composition allows the drug to traverse olfactory mucosa into brain tissue.

[0021] For some such embodiments, the critical temperature for gelation of a 50 pM solution of the ELP is less than about 34 °C and greater than about 20 °C in phosphate buffered saline solution. According to some embodiments, the ELP has a C-terminal or N-terminal cellpenetrating moiety. According to some embodiments, the cell-penetrating moiety is a polyarginine, wherein the number of arginines ranges from 4 to 20. According to some embodiments, the cell-penetrating moiety is a GRGDS sequence.

[0022] According to some embodiments, the drug is a polyphenol. According to some such embodiments, the drug is selected from the group consistion of vescalagin, curcumin, resveratrol, and combinations thereof.

[0023] According to some embodiments, a composition is disclosed including a drug and an ELP comprising multiple pentamer sequences of the form VPGXG, wherein X is any aminoacid except for proline. For some such embodiments, the ELP further includes a cell -penetrating moiety. For some such embodiments the composition has a critical temperature for gelation which is less than about 34 °C and greater than about 20 °C. For some such embodiments, X is selected from the group consisting of A, S, L, Y, F, and combinations thereof. For some such embodiments, the ratio of A to Y is from about 4:1 to about 2:3. For some embodiments, the total number of pentamer sequences is between about 10 and about 300. According to some such embodiments, the cell-penetrating moiety is a polyarginine, wherein the number of arginines ranges from 4 to 20. According to other embodiments, the cell-penetrating moiety is a GRGDS sequence.

[0024] For some embodiments, the amino acid sequence of the ELP has multiple repeat units of the sequence (VPGAG)2-(VPGXG)I-(VPGAG)2, wherein X is chosen from the amino acid group consisting of Y, F, S, L, and combinations thereof. According to some such embodiments, the multiple repeat units are selected from the group consisting of:[(VPGAG)2-(VPGYG)I-(VPGAG)2]24,[(VPGAG)2-(VPGFG)1-(VPGAG)2]24,[(VPGAG)2-(VPGSG)1-(VPGAG) 2]24,[(VPGAG)2-(VPGLG)I-(VPGAG) 2]24,[(VPGAG)2-(VPGLG)1-(VPGAG)2]24-[(VPGAG)2-(VPGYG)1-(VPGAG)2]24, [(VPGAG)2-(VPGYG)I-(VPGAG)2]24-[(VPGAG)2-(VPGLG)I-(VPGAG)2]24, [(VPGAG)2-(VPGYG)I-(VPGAG) 2]24-[(VPGAG)2-(VPGLG)I-(VPGAG)2]24-(R)IO, and combinations thereof.

[0025] According to some embodiments, a method is disclosed of drug delivery to the brain comprising delivering any of the above-disclosed ELP compositions to a patient intranasally. According to some embodiments, the composition is delivered for the treatment of a neurological disorder. For some embodiments, the neurologial disorder is Alzheimer’s disease.

[0026] In some embodiments of the present disclosure the use of thermo-gelling ELPs for IN applications using VSG as a model therapeutic for AD is pursued. ELPs were developed and screened to possess multiple properties including thermal gelation at nasal passage temperatures (34°C), non-covalent VSG binding, and VSG permeation through a nasal mucosa model. First, a library of tetramer ELPs was developed with different guest residue amino acids and tested for the gelation temperature via turbidimetry. This library was also screened for VSG drug bindingusing a “pull-down” assay where VSG+ELP is heated above the Tt and centrifuged. In this assay, VSG gets pulled into the resulting hydrogel pellet, and VSG concentration indicates the advantageous drug-binding ELP. Advantageous ELP from these tests was then used to develop a di-block octamer ELP library to stabilize microgel assembly and improve cell interactions for drug permeation through a nasal mucosa model. Specifically, an RPMI 2650 transwell nasal mucosa model was used to test VSG permeation with and without ELP. Some embodiments of the present disclosure utilize ELP guest residues for non-covalent complexing with a polyphenol and impellent thermo-gelling ELPs for IN applications.ELP Design and Synthesis:

[0027] The ELP monomer genes were designed using the criteria specific to the method known as recursive-directional ligation by gene termini modifications (GTMs-RDL) and inserted into pBluescript II SK (+) plasmids. Monomer genes encode 30 pps (pentapeptide sequence) repeat units where 80% of all guest residues contain alanine. The remaining 20% of guest residues were substituted with either tyrosine, phenylalanine, serine, or leucine amino acids. Using the GTMs-RDL restriction enzyme (RE) library (Bpil, Bmsl, Adel, and PagI), gene polymerization was carried out with these monomers 2-3 times to result in tetramer (120 pps) or octamer ELP genes (240 pps). Additionally, cell-interactive peptides (CIPs) were appended to the C-terminus of the octamer gene using Bpil and BamHI FastDigest® restriction enzymes. The CIPs include lOxR oligomers of 10 successive arginines, and GRGDS oliomers of the sequence glycine-arginine-glycine-aspartate-serine. The DNA oligomers used for CIP modification are shown in Table 1. All final ELP genes were sequenced through GENEWIZ to confirm the appropriate gene modification and were transferred to pET19b plasmids and transformed into BL21 (DE3) E. coli. Table 2 shows all final ELPs that were used in this study.Table 1. DNA oligomers used for appending the cell interactive moieties lOxR and GRGDS to the 3’ end of the octamer ELP gene, ELP-Y4L4. Bolded sequences indicate the overhang sequences present after annealing sense and antisense strands for ligating onto the ELP gene. Stop codons are italicized.10xR Oligos GRGDS Oligossense: g ggc cga egg ege cgt egg ege ega aga agg aga g ggc ggt cgt ggt gat tec tag tet teg tag tet tegantisense: g ate cga aga eta tot cot tot teg geg cog acg acg g ate cga aga eta gga ate acc acg cog teg accTable 2. The nomenclature of each ELP is shown along with its full amino acid sequence. Bolded symbols indicate the amino acids that vary between ELPs _Name Amino Acid SequenceELP-Y4 [(VPGAG)2-(VPGYG)1-(VPGAG)2]24ELP-F4 [(VPGAG)2-(VPG FG) 1 -(VPGAG)2]24ELP-S4 [(VPGAG)2-(VPGSG)I-(VPGAG)2]24ELP-L4 [(VPGAG)2-(VPG LG) 1 -(VPGAG)2]24ELP-L4Y4 [(VPGAG)2-(VPGLG)1-(VPGAG)2]24-[(VPGAG)2-(VPGYG)1-(VPGAG)2]24ELP-Y4L4 [(VPGAG)2-(VPGYG)1-(VPGAG)2]24-[(VPGAG)2-(VPGLG)1-(VPGAG)2]24ELP-Y4L4-WXR [(VPGAG)2-(VPGYG)1-(VPGAG)2]24-[(VPGAG)2-(VPGLG)1-(VPGAG)2]24-(R)1OELP phase transitions

[0028] Referring now to Fig. 1, each ELP shown in Table 2 was characterized by turbidimetry. As shown in Fig. 1 at low temperatures the ELP solution 10 is clear, but as the temperature is raised above Tt, gel formation results in a turbid ELP solution 15. The transition from solution to gel is readily monitored by following the absorbance at 350 (OD350). The results of such measurements are shown in Fig. 2 for heating curves (solid lines) and cooling curves (dashed lines) for ELP-F4 and ELP-Y4 in PBS buffer, as OD350 vs. temperature and as the derivative of OD350 vs. temperature. Shown in black for ELP-F4 are the forward transition 22 upon heating and the reverse transition 24 upon cooling. Shown in gray for ELP-Y4 are the forward transition 26 and the reverse transition 28. Under these solution conditions, the sol-geltransitions for ELP-S4 and ELP-L4 occurred at temperatures in excess of 50 °C and were not measured.

[0029] In order to compare the sol-gel tendencies of all tetramer species, turbidity measurements were also performed at higher salt concentrations, which shifts the transition temperatures to lower values. Without being bound by theory, because water is released upon gelation, this salt-dependent shift reflects the reduction in water activity at higher salt concentrations, which leads to a greater entropic contribution from water release during the gelation process.

[0030] Accordingly, the sol-gel transition was also monitored by turbidimetry for all tetramer species in solutions of PBS + 1.5 M NaCl. Under these conditions, all tetramers have transition temperatures below 50 °C. The results of all tetramer turbidity measurements are provided in Table 3, along with a summary of other experimental parameters determined for these molecules. Based on these experimental results, the order of tetramer ELP transition temperatures is ELP-S4 > ELP- L4 > ELP-F4 > ELP-Y4. This ranking agrees with the hydrophobicity scale. Moreover, cooling experiments (data not shown) indicate reversibility and re-solubilization of all tetramer ELPs.Table 3. Analyzed metrics for tetramer ELPs. Data is shown as mean ± standard deviation. PAGE stain quality indicates the labeling intensity of ELPs by a Coomassie stain and * indicates PBS buffer with added 1.5 M NaCl.Tetramer Yield Purity Calculated PAGE Stain Bioanalyzer PBS NaCl*(mg / 1L) (%AUC) MW(kDa) Quality MW(kDa) Tt (°C) Tt (°C) ELP-Y4 32.7 ± 8.4 94.42 ± 3.63 48.19 Good 81.13 ± 1.20 31.5 <4 ELP-F4 4.2 ± 1.5 98.66 ± 2.45 47.80 Good 62.33 ± 0.37 37.5 13.5 ELP-S4 6.2 ± 2.2 99.74 ± 0.32 46.36 Poor 100.2 ± 0.63 >50 45.5 ELP-L4 6.6 ± 1.4 89.43 ± 7.11 46.99 Poor 63.85 ± 2.75 >50 27.5

[0031] Turbidity transition curves were also obtained for octamer ELPs ELP-L4Y4, ELP-Y4L4, and ELP-Y4L4-IOXR, and the results are shown in Fig. 3 for ELP-L4Y4 (heating transition 32 and cooling transition 34) and ELP-Y4L4-IOXR (heating transition 36 and cooling transition38). These octamer constructs were designed to reduce bulk hydrogel formation by creating a diblock copolymer with a hydrophobic ELP core and a soluble ELP shell to reduce bulk hydrogel formation upon phase separation. The strategy was successful for all octamers, which showed no bulk hydrogel formation after 24 hours for a 50 pM solution of octamer. Upon phase separation, the octamer solutions formed spherical microparticles with diameters in the range of about 2 pm to about 10 pm.

[0032] Fig. 4 demonstrates the protective effect of VSG against oxidants. Cortical neurons were treated with 2.5 or 10 pM H2O2 with or without vescalagin and neuron viability was assessed via a PrestoBlue™ assay where a higher relative fluorescent unit (RFU) indicates better cell survival. As shown in this figure, in the absence of VSG, increasing doses of H2O2 kill cells and thus reduce measured fluorescence. However, as shown in Fig. 4, a therapeutically effective dose of 12.5 pM or 25 pM VSG provides substantial protection against the toxic effects of H2O2. Notably, these concentrations are well below the 50 pM range where VSG begins to show toxicity with primary cortical neurons (data not shown).Vescalagin Pull-Down Assays

[0033] Because turbidimetry analysis shows that all ELPs reversibly phase separate with added 1.5 M NaCl, the first pull-down assay for VSG was performed under these conditions such that all tetramer ELPs could be compared. VSG concentration was determined using a 2,2-diphenyl-l-picrylhydrazyl (DPPH) stable radical where DPPH was dissolved into EtOH at 1000 pM and mixed for 20 minutes. This stock DPPH solution was combined with aqueous VSG-containing samples in equimolar volumes for a final DPPH concentration of 500 pM. This concentration provided a linear calibration for VSG concentrations ranging from 10 to 50 pM.

[0034] The starting concentration of VSG was determined to be close to 50 pM regardless of the added tetramer ELP. This result shows that negligible DPPH is sequestered by tetramer ELPs at 1.5 M NaCl after immediate absorbance measurements. For all tetramer ELPs, a drop in VSG concentration in the supernatant was observed after hot centrifugation. This indicates that every tetramer ELP bound VSG upon phase separation. However, ELP-F4 shows the lowest concentration of VSG at 17.7 pM compared to all other ELPs. ELP-Y4 also shows a lower VSG concentration at 24.4 pM compared to 30.1 pM and 28.2 pM for ELP-S4 and ELP-L4, respectively. Measurements of the VSG concentration were also performed for the pellets.VSG is detected in the pellets for ELP-Y4 and ELP-F4, at 20.3 and 28.5 pM VSG, compared to 16.0 pM for ELP-S4 and 16.4 pM for ELP-L4. Taken together, these results imply that while all tetramer ELPs bind some level of VSG, phenyl-bearing amino acids can enhance the pull-down efficiency of polyphenols upon ELP phase separation.

[0035] As is evident from Table 3, of the tetramer ELPs, ELP-Y4 has the highest expression yield, and the most favorable transition temperature. Based on the pull-down experiments, ELP-Y4 also provides a therapeutic concentration within the gel phase.

[0036] Figs. 5-7 show the results of a pull-down assay in PBS buffer for the octameric species ELP-Y4L452 and ELP-Y4L4-IOXR 54, which demonstrates that VSG is sequestered into the gel phase under these conditions at a therapeutic dose.ELP and Nasal Carcinoma Cell Imaging

[0037] RPMI 2650 cells (ATCC) are squamous carcinoma cells originally obtained from the nasal septum of a cancer patient. Fig. 8 shows the distribution of RITC-labeled ELP- Y4L4 microgels 82 in the vicinity of RPMI 2650 cells 84. Fig. 9 shows (white dashed circles) that the RITC-labeled ELP-Y4L4-IOXR is taken up into the RPMI 2650 cells. And, Fig. 10 shows the self-assembly of RITC-labeled ELP-Y4L4-IOXR on the surface of the RPMI 2650 cells.Nasal Mucosa Model for Drug Permeation

[0038] RPMI 2650 human nasal epithelial cells were seeded into 0.4 pm pore 12-well transwell inserts at 170,000 cells per transwell in MEM (+NEAA) with 10% FBS, 1% GlutaMax™, and 1% penicillin-streptomycin. 1.5 ml of media was used in the basolateral compartments and 0.5 ml of media in the apical compartments with the cells, which denotes this culture period as the liquid-liquid culture (LLC) phase. Media was replaced in both compartments every 2 days for 8 days until cells were over 100% confluent in each transwell. On the eighth day, media from the apical compartments was completely removed to facilitate the RPMI production of mucus. This air-liquid interface (ALI) culture received 1.5 ml media changes to only the basolateral compartments every 1-2 days for 2 weeks (days 8-22). Any media in the apical compartment was aspirated during ALI culture, being careful not to disrupt the cell multilayer on the porous membrane of the transwell insert. After a week of ALI (-day15) mucus production can be visualized by a non-uniform “shiny” fdm developing on the cell layer. On the last day of culture, the film is nearly uniform and covers the entire transwell.

[0039] Mature (day 22) RPMI mucosa models were used to screen ELP + VSG treatments. All treatment groups were prepared in PBS with 50 pM VSG and 50 pM ELP. Three control groups were included in this study including 1) a PBS treatment control that has no drug or ELP effects, 2) a 50 pM VSG group lacking ELP, and 3) a VSG + 100% ELP-Y4L4 treatment group to compare effects due to cell interactive peptides in the experimental groups. After preparing all treatment solutions in PBS, all media was aspirated from the transwell models and the basolateral compartments received 500 pl of media. Subsequently, 500 pl of each treatment was added to the transwells and the plates were immediately placed in the cell culture incubator. After 30 minutes of incubation, all media was collected from the basolateral and apical compartments, taking care not to disrupt the cell multilayer. The basolateral compartment received 500 pl of fresh media and the plates were incubated for 10 hours, after which the basolateral media was collected again. VSG concentration in all collected samples was assessed using the DPPH assay.

[0040] Permeation from apical drug treatment to the basolateral compartment was assessed after 30 minutes using a DPPH assay, to mimic the dynamic mucous recycling in vivo. The results of this assay showed that VSG remains near its original concentration of 50 pM for ELP-Y4L4 and the VSG control, indicating that little to no VSG permeated through the cell membrane in 30 minutes. Notably, all ELP treatments containing lOxR cell-penetrating peptides pulled a statistically significant amount of VSG out of the apical compared to VSG only.However, very little VSG appears in the basolateral compartment for all treatments after 30 minutes indicating that the VSG missing in the apical compartment of the ELP-Y4L4-IOXR treatments is trapped at the cell membrane.

[0041] To mimic mucociliary clearance, apical treatments were removed after 30 minutes, and the basolateral media was replenished. Transwells were incubated for 10 hours, basolateral samples were collected again, and a DPPH assay was run to determine VSG concentration. Compared to the VSG only control, 100% ELP-Y4L4-IOXR had DPPH sequestering relative to the VSG-only control (average 17.3 vs 8.3 pM VSG, respectively). A toxicity assay was performed and showed no differences in toxicity among all control groups,indicating that the increased DPPH sequestering for 100% ELP-Y4L4-IOXR is due to VSG permeation and not to cell components released due to cell death or treatment toxicity.

[0042] Fig. 11 follows the time course of apical and basolateral concentrations of ELP -Y4L4 112 and ELP-Y4L4-IOXR 114. This figure demonstrates that while some RITC signal was detected for both octamer ELPs in the basolateral compartment after 10 hours, their concentrations were below 0.5 uM ELP. This indicates that the ELP acts more as a drug depot in the mucosa model rather than as a permeable drug carrier. Consequently, the results of this assay indicate that the detected VSG in the basolateral region after 10 hours of incubation is due to VSG release from gelled ELP at the cell surface rather than ELP carrying VSG through the membrane.

[0043] Taken together, these results show that ELP-Y4L4-IOXR not only captures the most VSG at the cell surface but enhances VSG permeation through the cell membrane compared to VSG alone or VSG combined with ELP lacking a cell-penetrating peptide.Together, this outcome indicates an advantage of using thermal-responsive ELPs with cellpenetrating peptides for polyphenol intranasal drug delivery.

[0044] Embodiments disclosed herein demonstrate the ability of an ELP to phase separate at nasal mucosa temperatures, bind VSG, and form hydrogel depots to enhance VSG permeation through a mucosa model. Some embodiments of the present disclosure include increasing the density of phenylalanine- or tyrosine-bearing pentapeptides to enhance VSG binding. In some embodiments, this involves adjusting the alanine: tyrosine ratio from about 4: 1 to about 3 :2 or about 2:3. This would lower the transition temperature and use shorter ELPs for phase separation at physiological temperatures. Shorter ELPs can also lead to micelle formation rather than hydrogel formation. Although micelles could reduce the benefit of bulk hydrogel formation for reducing mucociliary clearance, they can improve drug permeation due to their smaller size. Combining a lOxR tag with ELP micelles can help overcome the mucosal barrier more effectively. Additionally, shorter ELPs can reduce the risk of peptide accumulation in the brain with repeated doses, as smaller peptides are more easily cleared. Thus, increasing the tyrosine density can enhance drug binding, favor micelle formation, improve mucosal permeation, and minimize peptide accumulation.

[0045] This study focuses on facilitating ELP cell interactions via the lOxRtag. Many other cell-interactive peptides promote cell adhesion, cell uptake, and membrane immobilization.Some embodiments of the present disclosure are directed to screening additional cell interactive peptides with thermo-gelling ELPs (micelles or microgels) specifically in the context of mucus penetration during intranasal drug delivery. These cell-penetrating peptides can result in faster permeation or slower mucociliary clearance, ligand-binding motifs can also provide the advantage of targeting drug-bound ELPs to specific cellular receptors. Doing so can elicit intracellular uptake specifically by olfactory neurons instead of the epithelial cells, providing a more direct delivery route to the brain via the olfactory and trigeminal nerves.

[0046] Some embodiments of the present disclosure are directed to the delivery of other neuro-therapeutics to the brain via the IN route using ELPs. The ELPs used in this study can be complexed with other polyphenols such as curcumin, resveratrol, or other naturally derived plant tannins. ELPs can be appended with therapeutic proteins. Engineering ELPs to contain therapeutic moieties, e.g., neurotrophic factors, such as NGF and BDNF, that could facilitate neurite genesis, slow neurogeneration, etc., and undergo thermal gelation can provide a means of protein drug delivery to the brain via IN administration, which has yet to be demonstrated using an ELP substrate. Additionally, cationic peptide tags can be implemented onto the ELP to complex with gene therapies, which are becoming popular for CNS treatment via intranasal applications.

[0047] As used herein, the term “about” means within 10%.

[0048] Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions may be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

What is claimed is:

1. A composition comprising:an elastin-like polypeptide (ELP); anda drug,wherein the composition is configured such that intranasal delivery of the composition results in gelation and sequestration of the drug by the composition,wherein gelation of the composition and sequestration of the drug upon intranasal delivery of the composition allows the drug to traverse olfactory mucosa into brain tissue.

2. The composition according to claim 1, wherein the critical temperature for gelation of a 50 pM solution of the ELP in phosphate buffered saline solution is less than about 34 °C and greater than about 20 °C in phosphate buffered saline solution.

3. The composition according to either claim 1 or claim 2, wherein the ELP has a C-terminal or N-terminal cell-penetrating moiety.

4. The composition according to claim 3 wherein the cell-penetrating moiety is selected from the group consisting of a GRGDS sequence and a polyarginine for which the number of arginines ranges from 4 to 20.

5. The composition according to any one of claims 1-4, wherein the drug is a polyphenol.

6. The composition according to claim 5, wherein the polyphenol is selected from the group consisting of vescalagin, curcumin, resveratrol, and combinations thereof.

7. A composition comprising:an elastin-like peptide (ELP) comprising multiple pentamer sequences of the form VPGXG, and a cell-penetrating moiety, wherein X is any amino acid except for proline; and a drug,wherein the critical temperature for gelation of the composition is less than about 34 °C and greater than about 20 °C.

8. The composition according to claim 7, wherein X is selected from the group consisting of A, S, L, Y, F, and combinations thereof.

9. The composition according to claim 8, wherein the ratio of A to Y is from about 4: 1 to about 2:3.

10. The composition according to any one of claims 7-9, wherein the total number of pentamer sequences is between about 10 and about 300.

11. The composition according to any one of claims 7-10, wherein the cell penetrating moiety is a polyarginine, and wherein the number of arginines in the polyarginine ranges from 4 to 20.

12. The composition according to any one of claims 7-10, wherein the cell penetrating moiety is a GRGDS sequence.

13. The composition according to any one of claims 7-12, wherein the amino acid sequence has multiple repeat units of the sequence (VPGAG)2-(VPGXG)I-(VPGAG)2, wherein X is chosen from the amino acid group consisting of Y, F, S, L, and combinations thereof.

14. The composition according to claim 13, wherein the multiple repeat units are selected from the group consisting of:[(VPGAG)2-(VPGYG)I-(VPGAG)2]24,[(VPGAG)2-(VPGFG)1 -(VPGAG)2]24,[(VPGAG)2-(VPGSG)1 -(VPGAG)2]24,[(VPGAG)2-(VPGLG)1-(VPGAG)2]24,[(VPGAG)2-(VPGLG)I-(VPGAG)2]24-[(VPGAG)2-(VPGYG)I-(VPGAG)2]24,[(VPGAG)2-(VPGYG)I-(VPGAG)2]24-[(VPGAG)2-(VPGLG)I-(VPGAG)2]24,[(VPGAG)2-(VPGYG)I-(VPGAG)2]24-[(VPGAG)2-(VPGLG)I-(VPGAG)2]24-(R)IO, and combinations thereof.

15. The composition according to any one of claims 7-14, wherein the drug is a polyphenol.

16. The composition according to claim 15, wherein the polyphenol is selected from the group consisting of vescalagin, curcumin, resveratrol, and combinations thereof.

17. The composition according to claim 16, wherein the polyphenol is vescalagin.

18. A method of drug delivery to the brain comprising:delivering the composition of any one of claims 7-17 to a patient intranasally.

19. The method according to claim 18, wherein the composition is delivered for the treatment of a neurological disorder.

20. The method according to claim 19, wherein the neurological disorder is Alzheimer’s disease.

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