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Biomacromolecules
Biomacromolecules
bm
bomaf6
Biomacromolecules
1525-7797
1526-4602
American Chemical Society

39185801
10.1021/acs.biomac.4c00165
Article
Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach
https://orcid.org/0000-0002-4714-2618
Aayush Aayush
Darji Saloni
Estes Kiera M.
Yeh Emily
https://orcid.org/0000-0002-0746-1526
Thompson David H. *
Department of Chemistry & Purdue Institute for Cancer Research, Purdue University, Bindley Bioscience Center, West Lafayette, Indiana 47907, United States
* Email: davethom@purdue.edu.
26 08 2024
09 09 2024
25 9 57295744
05 02 2024
02 08 2024
02 08 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

non-viral nucleic acid delivery
nucleic acid encapsulation
elastin-like polypeptides
layer-by-layer formulation
targeted nanoparticles
bladder cancer therapeutics
National Cancer Institute 10.13039/100000054 CA23168 Purdue University 10.13039/100006377 NA document-id-old-9bm4c00165
document-id-new-14bm4c00165
ccc-price
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pmcIntroduction

Unraveling the human genome has opened many avenues to control and utilize genetic information to combat a vast array of diseases. Based upon this genetic information and the presence of driver mutations, nucleic acid (NA)-based therapies comprising siRNA, miRNA, mRNA, dsDNA, antisense oligonucleotides, and other NA constructs are being explored to expand the druggable sites of the genome.1 Realizing the vast potential of these approaches requires that the NA drug cross multiple physiological and cellular barriers to reach its site of action. Unfortunately, the high negative charge density, nuclease susceptibility, and rapid clearance of unmodified NA from circulation make access to intracellular sites of action difficult. Lessons from infectious agents like viruses and their design strategies for efficient packaging and transfer of genetic information have guided researchers toward designing nonviral delivery systems that protect and release their NA cargo within the cytoplasm of target cells.2,3

Viruses and virus-like particles encapsulate their NA cargo to prevent degradation and pathogen-associated molecular pattern recognition, deliver it to the host cell cytoplasm, and often display a tropism for specific cell types.4,5 Since these carriers have evolved to package a specific genome size, they typically have cargo size limits that challenge their adaptable manufacture and require surface residue engineering to escape immune surveillance6; they can also be challenging to reliably produce on large scale.7 Rational design of lipid-based nucleic acid nanoparticle (LNP) delivery systems was developed to address some of these limitations.5,8 The most successful formulations utilize ionizable cationic lipids to promote the endosomal release of the NA cargo,9 forming the basis of Alnylam’s Patisiran and the coronavirus vaccines developed by Bio-N-Tech/Pfizer and Moderna.10 Recent data show that N-oxidation of the ionizable lipid during manufacturing and/or storage leads to byproducts that react to form lipidated mRNA, rendering it untranslatable.11 This explains, in part, the low biological efficiency of LNP on a NA copy number basis, since they are estimated to only deliver 1% of the endocytosed NA cargo into the cytoplasm,12 although compositional13 and structural14 heterogeneity also may play a role. Cationic polymers are another large class of materials that have been developed to control the functionality and biophysical properties of transfection complexes.15,16 Polyethylenimine is a widely used polymeric transfection agent, however, it has very low plasmid delivery efficiency and high cytotoxicity at the loadings required for bioactivity.17−19 Polypeptide-based formulations generally have the advantage of facile incorporation of targeting ligands via solid-phase peptide synthesis or recombinant protein approaches, however, their homopolymers generally suffer from efficiency and cytotoxicity challenges.20

Elastin-like polypeptides (ELP) have emerged as a promising class of nonimmunogenic and biocompatible macromolecules that can be endowed with tunable stimuli-sensitive properties, making them suitable for various biomedical applications ranging from tissue engineering to gene delivery.21−25 Comprised of repeating VPGXG sequences (where X is any amino acid except proline), ELP typically possess very high hydropathies. Recent work from our lab has utilized this property for rapid purification using an organic solvent extraction/precipitation workflow with removal of host cell NA and lipopolysaccharides from ELP fusions expressed in Escherichia coli and retention of biological activity,26−29 including an ELP fusion capable of targeting human epidermal growth factor receptor (EGFR) ex vivo.28

This study describes our effort to develop an ELP-based carrier system capable of targeting bladder tumors to deliver NA therapeutics. Disease in the bladder is difficult to treat since intravesical instillation of chemo- or immunotherapies is confounded by the constant urine influx and voiding that limit target engagement times. For extended contact with the bladder along with improved tumor selectivity, both small molecule and macromolecular drugs have been shown to benefit from targeting approaches. Due to the high recurrence rate of bladder cancer, complete remission generally requires a combination of surgery and adjuvant chemo- and/or immunotherapy.30−32 The most common treatment regimen for nonmuscle invasive bladder cancer is the instillation of live attenuated Bacillus Calmette-Guerin (BCG) into the bladder after surgical resection of the detectable tumor. Co-localization of BCG mycobacteria with tumor tissue, enabled by the bacterial adhesin known as fibronectin attachment protein, leads to the immunogenic elimination of tumors near the mycobacterial binding site.33 Although this therapy has been the most substantial advancement in bladder cancer treatment, over 30% of patients are still unresponsive to this therapeutic modality.31,32 Additionally, since BCG is a live mycobacterium, there is an appreciable risk of systemic infection if the organism escapes the bladder compartment. A further complication of BCG therapy is a history of batch-to-batch variability, leading to manufacturing quality issues that contribute to a brittle BCG supply chain.34

We report a highly tunable and targeted delivery system that successfully delivers NA cargos ranging from 21bp siRNA to ∼6 kbp pDNA to bladder tumor cell lines. The nucleic acid cargo is first condensed with a decaarginine-β-cyclodextrin (CD-PLR10) conjugate and then wrapped with an ELP-epidermal growth factor (ELP-EGF) fusion protein coating. This layer-by-layer (LbL) approach35−39 not only provides shielding of excess positive charge on the oligocation-wrapped NA but also confers an ability to specifically target overexpressing EGFR tumor cells. Encouraged by the previous findings of scalable and rapid ELP purification by organic solvent extraction/precipitation,27 this targeted ELP NA carrier system is biomanufacturable, nonimmunogenic, and accommodates large variations in both the ELP nanoparticle vector components and NA cargos as required for the desired application. We also show that variation in the polycation type impacts transfection efficiency, likely due to differences in NA unpackaging efficiency and release from endosomal/lysosomal compartments, which is known to be a major hurdle for both lipid- and polymer-based delivery systems.

Materials and Methods

Polyion Complexation and LbL ELP:Nucleic Acid Nanoparticle (LENN) Formulation

The following stocks were prepared and used for all formulations unless stated otherwise: nucleic acid cargo (siRNA or pDNA) in RNAase-/LPS-free water (0.1 mg/mL) and polycation (CD-PLR10 or CD-PEI2.5k) in 18 MΩ H2O such that the concentration of positive charges (secondary amines in PEI2.5k and guanidinium groups in R10) was 10 mM. First, nucleic acid and polycations were vortex-mixed for 30 s at the desired N:P ratio and incubated at 20 °C for 30 min. N24-EGF (targeted) or N40 (untargeted) ELP was added to the polyion complexes with vortex mixing to form LENN. Varying amounts of ELP were added to produce defined molar ratios of the valine and proline residues in the LENN ELP layer. Formulation compositions are represented by suffixing the molar ratio and identity of the ELP, such that NP4–5xN24-EGF refers to a nanoparticle formulation where the amount of ELP added has a fivefold molar excess of valine + proline residues relative to CD-PLR10 (i.e., for each mole of CD used, the quantity of ELP solution added was calculated to achieve 5 mol of valines + prolines per mole of CD, such that for N24-EGF with a total of 72 valines + prolines in the (VPGVG)24 block of the ELP fusion, the calculated CD stoichiometry would be 72:14.4 or 5:1).

Agarose Gel Electrophoresis

Agarose gel electrophoresis was used to evaluate the nucleic acid complexation status in various formulations. In brief, agarose gels (2% for siRNA; 1% for pDNA) were cast by microwaving an appropriate amount of agarose in pH 7.4 Lithium Acetate Borate buffer (LAB buffer) until the solution turned clear and then cooled to touch before the addition of GelRed at a 1:10,000 dilution. The warm gel + dye solution was carefully poured to avoid any bubbles in the sample lanes in a gel holder with a comb of wells. Samples were diluted with a 6× sample loading buffer (without SDS). Nucleic acid complexation efficiency was determined by comparing the fluorescence signatures of GelRed-accessible nucleic acid binding relative to free nucleic acid as a positive control by using the same nucleic acid concentration and amount in each well. The gel was run at 200 V for 20 min and then imaged with a Bio-Rad Chemidoc Touch Imaging System.

Testing Stability of Formulations Using Heparin Challenge

Heparin was used to test the stability of the LENN formulations by agarose gel electrophoresis. The polyion complexes and LENN formulations were subjected to different ratios of heparin relative to the nucleic acid cargo (w/w) for 30 min. Two different heparin stocks were made to probe stability at both low and high molar equivalence heparin using equal volumes of the appropriate stock to mitigate dilution effects on the complexation/decomplexation behavior.

Atomic Force Microscopy

A Veeco MultiMode Atomic Force Microscope with a Nanoscope V Controller was used to obtain AFM images in tapping mode. AFM tips were 75 kHz with a 2.8 N/m force constant. Aqueous solutions of the nucleic acid formulations (0.5 μg/20 μL) were drop-cast onto mica and allowed to evaporate overnight before imaging.

TEM

Nucleic acid formulations were diluted 3–4-fold with 18 MΩ H2O before casting 3 μL of the solution onto the TEM grid. After 3–5 s, the grid was blotted, and then 3 μL of 1% uranyl acetate or phosphotungstic acid was added and incubated for 10–15 s before blotting away the excess and drying overnight in a desiccator before imaging. Images were acquired using a FEI Tecnai G2 20 electron microscope, equipped with a LaB6 source and a Gatan US1000 2K CCD, operating at 100–200 kV.

Dynamic Light Scattering

Particle size, size distributions, and ζ potential of the different formulations were determined by using a Malvern Zetasizer Nano ZS instrument. After formulation of 2 μg of nucleic acids into polyion complexes or LENN, 700 μL of 10 mM HEPES + 10 mM NaCl solution (pH = 7) was added for size and ζ measurements. All measurements were taken in triplicate.

Cell Culture

Mycoplasma-free T24 human (procured from ATCC) and MB49 mouse urothelial carcinoma (gift from Prof. Timothy Ratliff, Purdue Institute for Cancer Research) cell lines were used to evaluate the performance of various NA complexes. T24 cells were cultured in McCoy’s medium supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. MB49 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin under the same conditions as the T24 cells.

Transfection to Bladder Tumor Cell Lines and Measurement of Toxicity

Cells were seeded at a density of 50,000 cells/well in 12-well plates overnight in serum-containing media and allowed to attain 30–50% confluency. Serum-containing media was replaced with serum-free media 2–3 h before the addition of the NA formulations and incubation at 37 °C and 5% CO2 for 5 h. The NA treatments were then removed, and the cells were supplemented with serum-containing complete media and incubated for varying times before measuring toxicity using the CellTiter 96 AQueous assay as per the recommended protocol. Typical cell treatment amounts were 2 μg for pDNA and 0.54 μg for siRNA.

Cell Binding Assessment by Flow Cytometry

The nanoparticles were formulated with Cy5.5-conjugated ELP and the cells were prepared for transfection as described above. For serum competition studies, FBS was added to the cells to obtain a 20% (v/v) solution before the addition of the NA formulations. After incubating the NA complexes with cells for different time periods at 37 °C under 5% CO2, cells were washed with 1X PBS, followed by resuspension in 1 mL flow cytometry staining buffer (1% BSA and 0.1% NaN3) and filtered through tubes with a cell strainer cap to remove multicellular clusters. Cell slurries from various treatments were evaluated using a BD LSRFortessa cell analyzer using the APC-Alexa 700 channel (λex: 640 nm/λem: 710 nm), and the data were processed using FCS Express 7.

Cell Binding and Internalization Monitored by Confocal Microscopy

Poly-l-lysine coated slides (6-well plates) were seeded with 150,000 cells/well and incubated in serum-containing media until ∼50% confluency was reached. Time-based internalization studies were performed for the same conditions and concentrations as those in the binding studies. Post-treatment, cells were washed with 1× PBS and then incubated in 2% paraformaldehyde for 10–15 min (5 min when analyzing GFP expression), followed by 2 more washes with 1× PBS. The coverslips were then mounted on glass slides using ibidi mounting media (with or without DAPI) and analyzed with a Nikon AIR-MP microscope using the fluorescence channels (DAPI, TRITC, FITC, Cy5.5) relevant for the particular experiment.

Fluorescence Studies Using 6-(p-Toluidino)-2-naphthalenesulfonic Acid (TNS)

siRNA and CD-PLR10 polyplexes were prepared and distributed into 384 wells under various conditions, 20 μM TNS was added, and the solutions were mixed by pipetting to give a TNS concentration of 10 μM (final concentrations of CD-PLR10 ≈0.1 mM and siRNA ≈ 0.05 μg/μL). After 45 min at 20 °C, the fluorescence intensity was monitored [ex: 320 nm; em: 500 nm] before the addition of N24-EGF to give a range of concentrations (1x- 0.70 μM, 2x- 1.41 μM, 3x- 2.05 μM, 4x- 2.66 μM, 5x- 3.23 μM) and pipet-mixed before monitoring the fluorescence intensity again.

Results and Discussion

Nucleic Acid Encapsulation Using a LbL Approach

The nucleic acids were first electrostatically condensed by vortex mixing them with a polycationic β-cyclodextrin (CD-PEI2.5k or CD-PLR10, Supporting Information Schemes S2 and S3, respectively) conjugate at varying N:P ratios. These polyion complexes were then incubated with a solution of ELP (N40 or N24-EGF fusion protein) to form LbL ELP:nucleic acid nanoparticles, LENN (Figure 1). Alonso et al. have shown that the valine and proline residues of ELP engage in weak hydrophobic interactions with β-cyclodextrin.40 We reasoned that a large number of dynamic interactions between the nonpolar ELP prosthetic groups and the hydrophobic β-cyclodextrin cavity would lead to further condensation into stable LENN complexes due to the high cooperativity of multiple host:guest interactions. We also anticipated that it would provide a wide spectrum of control over the nanoparticle composition and assembly/disassembly kinetics that are critical for endosomal escape of the cargo. NA wrapping by ELP serves two additional important functions in the bladder cancer therapeutics context: [1] shielding the excess positive charge on the electrostatically condensed polyplexes to limit nonspecific adsorption to the glycans-rich bladder lumen; and [2] incorporation of the EGF targeting ligand to promote tumor selectivity since EGFR is known to be upregulated in 74% of bladder tumor cases.30,41,42

Figure 1 Layer-by-layer formation of elastin-like polypeptide:nucleic acid nanoparticles, LENN. In the first step, the nucleic acids (siRNA or pDNA) are electrostatically condensed by complexation with polycations (decaarginine- or 2.5 kDa polyethylenimine-modified β-cyclodextrin) to form polyion complexes. These initially formed particles are then stabilized with an ELP coating (N24-EGF to target epidermal growth factor receptor, EGFR, and/or N40 as an untargeted construct) through host:guest interactions between the cyclodextrin cavities and the hydrophobic prosthetic groups of the ELP.

This highly adaptable approach offers the opportunity to tune LENN properties through simple modifications of the formulation process to enable optimal cell binding, internalization, and intracellular cargo release across a wide range of NA cargo sizes. Table 1 summarizes some of the key distinctions among viral vectors, LNP, and the LENN-based approach. Three key drivers for pursuing an ELP-based approach are the [1] biocompatibility and nonimmunogenicity of ELP compared to viral vectors, [2] obviation of the chemical instability found in lipid formulations using ionizable lipids,11 and [3] biologic expression of a carrier system that is flexible in its ability to target and deliver nucleic acid cargo of differing sizes.

Table 1 Comparison between Viral Systems, LNP, and LENN for Nucleic Acid Delivery

vector type	virus	LNP	LENN	
properties	condensation element	nucleocapsid proteins	protonated ionizable lipid self-assembly	cyclodextrin-polycation	
stealth element	 	PEG (polydisperse synthetic polymer)	ELP (polypeptide of uniform sequence)	
targeting element	receptor-binding domains of capsid proteins	receptor-specific ligands (in some cases)	receptor-specific ligand	
release element	fusogenic peptides	endosomal membrane permeabilization mediated by ionizable lipid phase transition	contact of endosomal membrane lipids with oligopeptide and β-CD cavity	
cargo size	≤4 kb DNA or RNA	siRNA-pDNA	siRNA-pDNA	
liabilities	immunogenic	physical & chemical instability	protein stability	

Biophysical Characterization of Polyion Complexes and LENN

siRNA Encapsulation: Size and Shape

Transmission electron microscopy (TEM) images were collected after staining the LENN complexes with 1% uranyl acetate. The initial CD-PLR10:siRNA polyion complexes appear as a mixture of spheres and ellipsoids with diameters of ∼50 nm. After the addition of N24-EGF to the polyion complexes, the LENN formulations displayed a mixture of 200–300 nm aggregates that appeared as 2-D networks of smaller assemblies and spherical 50 nm particles (Figure 2A, Supporting Information Figure S2).

Figure 2 TEM and AFM characterization of N24-EGF, siRNA:CD-PLR10polyion complexes and siRNA:CD-PLR10:ELP LENN.(A) TEM images using 1% uranyl acetate as negative stain. (B) AFM data were collected on cleaved mica surfaces. N24-EGF: native N24-EGF fusion protein without other formulation components; NP2: CD-PLR10:siRNA polyion complexes with at N:P = 2; NP2–5x N24-EGF: N24-EGF:CD-PLR10:siRNA LENN with a fivefold molar excess of hydrophobic prosthetic groups (prolines + valines) with respect to β-CD cavities at N:P = 2; NP4: CD-PLR10:siRNA polyion complexes at N:P = 4; NP4–5x N24-EGF: N24-EGF:CD-PLR10:siRNA LENN with a fivefold molar excess of hydrophobic prothetic groups (prolines + valines) with respect to β-CD at N/P = 4.

Since globular and network structures were also observed for N24-EGF alone (Figure 2A), we infer from these observations that the “diblock” amphipathic N24-EGF sequence may be behaving similarly to other diblock ELP designs that have shown similar aggregation behavior.43,44 The sizes and size distributions of nanoparticles observed by TEM were also corroborated by AFM and DLS (Figure 2B and Table 2; Supporting Information Figure S3 and Table S1). Bravo-Anaya et al. have reported similar structures in their AFM experiments with cationic ELP encapsulated plasmids and described a process wherein the ELP first condenses around the nucleic acid before forming network-like structures with other condensates.45

Table 2 Observed Diameters and Zeta Potentials of Polyion Complexes and LENN as Determined by Dynamic Light Scattering (Average of 3 Samples)

formulation	Dave (nm)	ζave (mV)	
NP2	159	–0.04	
NP2–5x N24-EGF	136	–0.04	
NP4	116	9.8	
NP4–5x N24-EGF	119	1.7	

These findings suggest that the ELP can self-associate, thus highlighting the importance of striking the right stoichiometric balance of ELP:cationic β-cyclodextrin to form stable LENN complexes via binding of the ELP hydrophobic residues to the clustered apolar cyclodextrin cavities instead of self-associating to generate network structures.

pDNA Encapsulation: Size and Shape

The pDNA LENN complexes were observed to be ∼80 nm and more uniformly spherical than the siRNA complexes at higher N:P ratios as reported for other pDNA delivery systems.46,47 The addition of N24-EGF did not lead to any further observable compaction; however, the particle size distribution became broader, ranging from 30 to 200 nm, regardless of the N:P ratio (Supporting Information Figure S5) or polyion complex:ELP ratio employed (Figure 3).

Figure 3 TEM characterization of pDNA:CD-PLR10polyion complexes and pDNA:CD-PLR10:N24-EGF LENN complexes. The polyion complexes and LENN particles were cast onto TEM grids and stained with 1% UA before imaging. NP10: pDNA:CD-PLR10 polyion complexes at N:P = 10; NP10–5x N24-EGF: pDNA:CD-PLR10:N24-EGF LENN with a fivefold molar excess of hydrophobic prosthetic groups (prolines + valines) with respect to β-CD cavities at N:P = 10.

Nucleic Acid Encapsulation: Mechanistic Insight into Formulation Behavior

TNS Fluorescence Changes

TNS is a solvatochromic fluorophore that becomes highly fluorescent in hydrophobic media.48 We performed a series of experiments with TNS to probe the LbL assembly behavior during LENN complex formation. When TNS was added to unmodified β-cyclodextrin, CD-PLR10, and NP4 siRNA:CD-PLR10 polyion complexes, the fluorescence intensity increased compared to the aqueous controls at the same probe concentration, with the highest observed increases observed for CD-PLR10 and NP4 siRNA:CD-PLR10 polyion complexes (Supporting Information Figure S6A). We attribute these observations to the combined effect of TNS host:guest inclusion within the hydrophobic β-cyclodextrin cavity and enhanced binding due to ion pairing of the TNS sulfonate with the CD-PLR10 polycationic ponytail.49 After the addition of N24-EGF, the change in TNS fluorescence between t = 0 min and t = 45 min of incubation was positively correlated with the amount of ELP added (Figure 4A; Supporting Information Figure S6B). We also observed that the magnitude of the TNS fluorescence enhancement was smaller when the ELP was mixed with polyion complexes (Figure 4B; Supporting Information Figure S6C–G). Taken together, we infer from these observations that TNS is sensing an increased hydrophobic environment in the vicinity of the probe due to the cooperative binding of the ELP prosthetic groups to preformed polyion complexes.

Figure 4 6-p-Toluidinylnaphthalene-2-sulfonate (TNS) fluorescence-based assays: The concentrations of components were kept similar across different conditions; the polyplexes were prepared then distributed into different wells followed by addition of TNS and stabilization for 45 min (see also Supporting Information Figure S6A,B); wells with just ELP were prefilled with H2O followed by TNS addition. (A) Change in TNS fluorescence 45 min post ELP addition to H2O + TNS wells compared to right before the addition. (B) Change in TNS fluorescence 45 min post ELP addition to NP4 + TNS wells compared to the right before addition. Statistical differences were calculated by ANOVA (p < 0.05) followed by Tukey’s multiple comparisons (p < 0.05) using GraphPad Prism v5.0, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Agarose Gel Analysis

Both siRNA and pDNA formed stable complexes with CD-PLR10 and CD-PEI2.5k (Figure 5). CD-PLR10 formed more stable polyion complexes than did CD-PEI2.5k. The stability of the polyion complexes and LENN toward heparin challenge was also tested as a function of the encapsulated NA:heparin ratio. As the data in Figure 5D,E show, LBL coating of the polyion complex with ELP imparts an enhanced resistance toward LENN disassembly in the presence of heparin. We infer from these findings that the host:guest interactions between the hydrophobic cavities of CD-PLR10 and the ELP prosthetic groups confer a charge shielding effect on the polyion complexes, consistent with the observed reduction in ζ potential for the complexes (Table 2, NP4 vs NP4–5x N24-EGF).

Figure 5 Encapsulation efficiency of siRNA and pDNA within different LENN complex formulations and their stability toward heparin challenge. Agarose gel characterization of polyion complexes (siRNA:CD-PLR10 and siRNA:CD-PEI2.5k) at various N:P ratios and LENN complexes (siRNA:CD-PLR10:N24-EGF and CD-PEI2.5k:N24-EGF) at 5x N24-EGF before and after heparin challenges of varying ratios; 100 ng of siRNA loaded in each lane. (A) siRNA polyion complexes (siRNA:CD-PLR2.5k and siRNA:CD-PLR10) at varying N:P ratios, with Lanes 1–5 showing CD-PEI2.5k complexes and Lanes 6–10 showing CD-PLR10 complexes. Lane 1, NP2; Lane 2, NP4; Lane 3, NP6; Lane 4, NP8; Lane 5, NP10; Lane 6, NP2; Lane 7, NP4; Lane 8, NP6; Lane 9, NP8; Lane 10, NP10; Lane 11, siRNA alone. (B) siRNA LENN formulations (siRNA:CD-PEI2.5k-5xN24-EGF and siRNA:CD-PLR10-5xN24-EGF) were obtained at varying N:P ratios. Lane 1, CD-PEI2.5k:5xN24-EGF at NP2; Lane 2, CD-PLR10:5xN24-EGF at NP2; Lane 3, CD-PEI2.5k:5xN24-EGF at NP4; Lane 4, CD-PLR10:5xN24-EGF at NP4; Lane 5, CD-PEI2.5k:5xN24-EGF at NP6; Lane 6, CD-PLR10:5xN24-EGF at NP6; Lane 7, CD-PEI2.5k:5xN24-EGF at NP8; Lane 8, CD-PLR10:5xN24-EGF at NP8; Lane 9, CD-PEI2.5k:5xN24-EGF at NP10; Lane 10, CD-PLR10:5xN24-EGF at NP10. (C–E) Agarose gel characterization of polyion complexes (pDNA:CD-PLR10 and pDNA:CD-PEI2.5k) and LENN complexes (pDNA:CD-PLR10:N24-EGF and pDNA:CD-PEI2.5k:N24-EGF). (C) Cy5.5-N24-EGF was used in these formulations, and 300 ng of pDNA was loaded in each lane. Lane 1, pDNA:CD-PLR10:5xN24-EGF at NP8; Lane 2, pDNA:CD-PLR10:5xN40 at NP8; Lane 3, pDNA; Lane 4, Cy5.5-N24-EGF; Lane 5, pDNA:CD-PLR10:Cy5.5-N24-EGF at NP8 (Lanes 4 and 5 were visualized in the near-infrared emission channel). (D) Polyion complexes (pDNA:CD-PLR10 and CD-PEI2.5k) and pDNA:CD-PLR10:N24-EGF LENN complexes. Note that 2xh and 5xh refer to incubation with heparin 2 times and 5 times w/w of pDNA, respectively. (E) Stability of pDNA:CD-PEI2.5k polyion complexes and pDNA:CD-PEI2.5k:ELP LENN toward heparin challenge (300 ng of pDNA). Lane 1, pDNA; Lane 2, pDNA:CD-PEI2.5k at NP7; Lane 3, pDNA:CD-PEI2.5k:5xN40 at NP7; Lane 4, pDNA:CD-PEI2.5k:5x N24-EGF at NP7; Lane 5, pDNA:CD-PEI2.5k at NP7 with 0.1 × h; Lane 6, pDNA:CD-PEI2.5k:5xN40 at NP7 with 0.1 × h; Lane 7, pDNA:CD-PEI2.5k:5xN24-EGF at NP7 with 0.1 × h; Lane 8, pDNA:CD-PEI2.5k at NP7 with 2 × h; Lane 9, pDNA:CD-PEI2.5k:5xN40 at NP7 with 2 × h; Lane 10, pDNA:CD-PEI2.5k:5xN24-EGF at NP7 with 2 × h.

Although the heparin challenge is a commonly employed stability test in NA-based formulations, it serves a particularly important role in testing formulations for bladder cancer therapeutics by mimicking the bladder lumen environment and its dense coverage with negatively charged glycans. Further support for ELP wrapping around the polyion complexes is provided by the difference in migration behavior for the free and complex-bound forms of Cy5.5-N24-EGF in an electric field (Figure 5C, Lane 4). The net negative charge within the EGF domain of N24-EGF promotes migration toward the cathode under an applied potential, whereas this movement is abrogated when the pDNA:CD-PEI2.5k polyion complexes are wrapped with N24-EGF via LBL deposition. Encouraged by these findings of near-neutral ζ potential and resistance to heparin challenge, we proceeded with cell-based studies to evaluate the biological performance of the LENN system.

Biochemical Characterization of NA:CD-PLR10:ELP LENN

Human and Murine Bladder Cancer Cells (T24 & MB49) Bind and Internalize siRNA LENN

Cy5.5-conjugated N24-EGF (targeted) and N40 (nontargeted) were used to form siRNA LENN for flow cytometric analysis of their interaction with T24 (human) and MB49 (murine) bladder cancer cells. We observed that Cy5.5-N24-EGF containing LENN produced a strong cell-associated fluorescence in both cell lines that increased with incubation time (Figure 6A,F,G; Supporting Information Figures S7 and S8). Conversely, little to no Cy5.5 fluorescence was observed in T24 cells incubated with Cy5.5-N40 LENN at NP4 for up to 5 h. We concluded from these findings that the targeted formulations had enhanced cell association through engagement of EGF receptors (EGFR) on the tumor cell surface. Further support for this hypothesis is provided by EGFR blockade studies using serum-supplemented media before treatment with the LENN formulations that produced a reduction in cell-associated fluorescence due to competition for EGFR by LENN and the free EGF present in the media (Figure 6B; Supporting Information Figures S7 and S8). The cell-associated fluorescence stagnates by 2 h in T24, a finding that we attribute to maximum binding and internalization by that time point (Supporting Information Figure S7). It is important to note that 2 h is the practical therapeutic window for agents administered by intravesical infusion; washout through micturition typically occurs beyond this time window. It should also be noted that MB49 is a higher EGFR-expressing cell line than T24,28 thus accounting for the higher cell-associated fluorescence levels observed upon treatment of MB49 cells with N24-EGF containing formulations (Supporting Information Figures S7 and S8). Formulations prepared at lower NP ratios, but the same ELP ratio, also showed lower cell-associated fluorescence, likely due to the lability of these complexes that impeded their efficient binding and/or internalization (e.g., T24: NP2–5x N24-EGF in Supporting Information Figure S7 vs NP4–5x N24-EGF in Figure 6A; MB49: NP2–5x N24-EGF and NP4–5x N24-EGF in Figure 6F vs G).

Figure 6 T24 & MB49 bladder tumor cell association with siRNA:CD-PLR10:ELP LENN as a function of time and LENN composition. Flow cytometry analyses of cell-associated fluorescence after incubation with different Cy5.5-ELP LENN (N40, untargeted; N24-EGF, targeted); the ELP content (valine + proline residues) was 5 times the molar ratio of CD-PLR10. (A) Temporal changes in cell association of LENN (binding + internalization): NP4–5x ELP (Cy5.5-N24-EGF or Cy5.5-N40). (B) Addition of serum-containing free EGF before addition of NP4–5x N24-EGF led to a reduction in cell-associated fluorescence compared with treatments with NP4–5x N24-EGF LENN in serum-free media. (C) Temporal changes in T24 cell association after incubating with free Cy5.5-N24-EGF and Cy5.5-N40 at the same concentration used in the formulations shown in Figure A. (D) Cell-associated fluorescence of NP4–5x ELP LENN at 1 and 3 h, where ELP = a physical mixture of Cy5.5-N40 and Cy5.5-N24-EGF during the layer-by-layer deposition step, with ratios ranging from 0:5 to 5:0 such that the total number of moles of (proline + valines) is 5 times that of CD-PLR10 in each case. The total number of live cells counted for each condition was kept similar in Figures D,E. (E) Changes in cell-associated fluorescence upon treatment of MB49 cells with NP2–5x ELP (N24-EGF or N40) for 0.5, 1, and 3 h. (F) Changes in MB49 cell-associated fluorescence upon treatment with NP4–5x ELP (N24-EGF or N40) for 0.5 1, and 3 h.

Free ELP and ELP-wrapped siRNA:CD-PLR10 LENN complexes have similar cell-associated fluorescence, at least up to 1 h (Figure 6C vs A,B). Previous work showed that serum-based competition occurred within 30 min when the free N24-EGF peptide was incubated with T24 cells, unlike N24-EGF LENN formulations that display greater avidity for T24 cells in the presence of serum (Supporting Information Figure S7). We interpret this resistance to serum competition at 30 min as arising from two potential origins: [1] reduced availability of free receptors on the T24 cell surface due to multivalent engagement of EGFR by LENN that presents multiple copies of spatially restricted EGF; and [2] EGF present in the culture medium may stimulate micropinocytosis, a secondary mechanism of EGFR uptake, leading to the observed initial increase in fluorescence upon addition of serum as occurs for other tumor cell lines.50

EGF titrations were then performed to evaluate the relationship between cell interaction efficiencies as a function of N24-EGF content within blended LENN formulations (Figures 6D,E). After 1 h of incubation, we observed increased cell-associated fluorescence as the EGF content in the LENN nanoparticles increased from 0 to 100% N24-EGF (Figure 6D). Interestingly, after 3 h incubation, LENN formulated with 100% N24-EGF showed less enhancement in cell-associated fluorescence compared to LENN with lower N24-EGF loading (c.f., Figure 6E, 5:0 vs 4:1 and 3:2 N24-EGF). Since EGFR is internalized primarily through clathrin-mediated endocytosis and recent mechanistic studies on the effect of nanoparticle elasticity on tumor uptake show that stiff spherical particles are more readily internalized by clathrin-mediated pathways than deformable nanoparticles,51,52 the increased cell-associated fluorescence with increasing EGF content may suggest that multivalent engagement of EGFR may serve to rigidify the LENN. In that scenario, enhanced uptake and EGFR recycling would be expected until receptor saturation occurs.53,54

Confocal microscopy images of T24 cells were collected after they were incubated with dual-labeled LENN formulated with Cy5.5-labeled ELP and Rhodamine-siRNA to determine the time-dependent distribution of LENN components after binding (Figure 7A). Our data show that EGF-targeted LENNs are internalized within 30 min and to a greater extent than either Lipofectamine or the polyion complexes lacking an ELP LbL coating. The images in Figure 7B also reveal an initial loss of colocalized Cy5.5 and Rh signals within 1 h, a finding that we interpret as the onset of LENN dissociation into different cellular compartments. Taken together, these findings show the general trend of increased dispersion of both the Cy5.5 and Rhodamine signals with increasing incubation time, such that the targeted Cy5.5-N24-EGF LENN formulations showed greater uptake and intracellular distribution than polyionic complexes at NP = 2 or Lipofectamine controls.

Figure 7 Time-dependent intracellular fate of LENN in T24 human bladder tumor cells. Confocal analyses of particles formulated with rhodamine labeled siRNA (red), CD-PLR10, Cy5.5-ELP (green), and DAPI-labeled nuclei (blue). T24 cells were incubated with NP2–5x Cy5.5-N24-EGF, Lipofectamine, and NP2 (no Cy5.5 label) for 30, 60, or 120 min before washing, fixing, and imaging. Note: Any applied image correction had been normalized across all of the conditions.

Bladder Cancer Cells Bind and Internalize pDNA LENN Complexes

Confocal images were collected after incubating MB49 cells with dual labeled LENN formulated using either Lipofectamine, nontargeted Cy5.5-N40, or targeted Cy5.5-N24-EGF to encapsulate FITC-modified pDNA (Figure 8). We observed that the targeted LENN performed better than the commercial transfection standard, with respect to tumor association and internalization efficiency. No significant internalization was detected in cells treated with the untargeted LENN, underscoring the importance of EGF in the formulation for the engagement and uptake of the LENN vector.

Figure 8 Binding and internalization of pDNA:CD-PLR10:ELP LENN by MB49 cells. Confocal analyses of particle association with MB49 cells after 3 h of incubation. N24-EGF and N40 LENN formulations were prepared from FITC-labeled pDNA (green), CD-PLR10, and Cy5.5-labeled ELP (magenta). Nuclei were stained with DAPI (blue). LENN formulated with untargeted Cy5.5-N40 displayed very weak cellular fluorescence for either the Cy5.5-ELP or FITC-pDNA associated fluorescence channels. LENN formulated with N24-EGF showed extensive cellular association, similar to that of Lipofectamine. Note: Any applied image correction had been normalized across all the conditions.

We also observed diffuse fluorescence spread over the cells after incubating T24 and MB49 with just Cy5.5-N24-EGF, consistent with previous observations in Cy5.5-N24-EGF studies.28 Moreover, the binding kinetics of these N24-EGF LENN within the first 30 min appears similar to the free peptide binding rates. We infer from these observations that the cell-association avidity of N24-EGF is retained even when present in nanoparticle formulations, thus setting the stage for testing the biological efficacy of the LENN delivery systems in mammalian tumor cells.

Biological Performance of Nucleic Acid:CD-PLR:ELP LENN

siRNA LENN Mediated Silencing of Hypoxia-Inducible Factor-1α (HIF-1α)

In an effort to evaluate the NA-delivery efficiency of the LENN system, we focused on HIF-1α siRNA knockdown due to the importance of this target in bladder cancer recurrence.55 Several recent studies have revealed the negative impact of hypoxia-inducible factors (HIF) expression in urothelial carcinoma and its positive correlation with chemotherapy resistance, immunotherapy failures, and resistance to BCG therapy.55−57 HIF-1α is a transcription factor that is stably expressed in hypoxic environments. Bladder tumors are a prime example of a tissue type that possesses hypoxic microenvironmental niches and a limited glucose supply due to poor vascularization. The combination of the two environmental stressors generally leads to aggressive behaviors in tumors through HIF-1α activation of various pro-tumorigenic cellular pathways, ultimately increasing tumor plasticity and functional heterogeneity.58 This leads to the activation of “novel” metabolic escape pathways, higher invasive capacity, resistance to cytotoxicity, increase in checkpoint inhibitor expression, and other immune suppression pathways.59,60 By impacting HIF-1α expression, we sought to target a crucial driver in the evolutionary progression of cancer and its dissemination induced by the bladder tumor microenvironment.

EGFR overexpression has been reported in 74% of bladder cancer patients.61 It plays a role in many pre- and pro- oncogenic pathways responsible for cell survival, repair, migration, invasion, and cell differentiation.62 Many therapeutic approaches have been developed to target the suppression of EGFR function with mixed success. Many of these have focused on kinase inhibition, however, upon development of tumor resistance toward these therapies, it was discovered that even in the absence of its kinase functions, EGFR participates in pro-survival functions in tumors.63 From a drug delivery perspective, the higher abundance of EGFR on tumors (high and low grades) compared to normal cells and its capacity for receptor-mediated endocytosis upon ligand binding make it an attractive route for intracellular delivery of nucleic acid loaded LENN bearing EGF targeting ligands.

We tested for abrogated HIF-1α mRNA expression using RT-PCR after incubating the cells with various formulations of siRNA:CD-PLR10:ELP LENN. In general, the targeted siRNA LENN complexes produced greater gene silencing than the nontargeted siRNA LENN or siRNA:CD-PLR10 polyion complexes lacking the N24-EGF coating (Figure 9). These findings are consistent with an enhanced delivery of functional siRNA to the cytoplasm of bladder tumor cells mediated by the EGFR uptake of the EGF-bearing LENN complexes.

Figure 9 HIF-1α silencing in T24 cells by siRNA:CD-PLR10:ELP LENN. Relative expression levels of HIF-1α at 70 h post-transfection as determined by RT-PCR. YC-1 is a small molecule inhibitor of HIF-1α. The formulations with ELP were made using a 5x ELP:CD ratio. All the conditions have been normalized against normoxia and scaled by their exerted toxicity (Supporting Information Figure S10). Statistical differences were calculated by ANOVA (p < 0.05) followed by Tukey’s multiple comparisons (p < 0.05) using GraphPad Prism v5.0, where * = p < 0.05, ** = p < 0.01.

Transfection of Murine Bladder Cancer (MB49) and Macrophage (RAW264.7) Cells by pDNA LENN

As a further test of the adaptability of the LENN system, we sought to deliver a large pDNA payload and test for the gene expression efficiency. MB49 cells were treated with pDNA:CD-PLR10:ELP LENN and evaluated for GFP expression at 48 h post-transfection (Figure 10). Formulations with N24-EGF produced maximum GFP expression compared to Lipofectamine and polyion complexes lacking an ELP LbL coating. Cytotoxicity was higher in the cases of cells treated with Lipofectamine and polyion complexes, as suggested by lower overall cell numbers in those wells (Figure 10A). High GFP expression in MB49 cells was also observed for pDNA CD-PEI2.5k LENN formulations compared to their polyion complexes lacking an ELP coating or Lipofectamine controls (Supporting Information Figure S11).

Figure 10 Transfection of pDNA:CD-PEI2.5k:ELP LENN and polyion complexes. Confocal microscopy analysis of GFP expression 48 h post-transfection with various pDNA:CD-PEI2.5k formulations. (A & C) MB49 cells. (B & D) RAW264.7 cells. Lipofectamine was used as a positive control for transfection. Note: Any applied image correction had been normalized across all the conditions. Microscopy images of LENN complexes and Lipofectamine were analyzed using ImageJ software and the grayscale intensities used to derive average fluorescence intensities over the areas of interest. Area averaged fluorescein intensities were divided by the area averaged DAPI intensities to normalize the fluorescence intensities across different treatments. The statistical differences were calculated by ANOVA (p < 0.05) followed by Tukey’s multiple comparisons (p < 0.05) using GraphPad Prism v5.0, where *= p < 0.05, ** = p < 0.01, and *** = p < 0.001.

In bladder cancer, there is generally a close spatial interaction between tumor-associated macrophages and tumors. Our next experiments used RAW264.7 cells to mimic the particle uptake process by tumor-resident macrophages. pDNA:CD-PLR10:ELP LENN and pDNA:CD-PLR10 complexes were more successful at plasmid transfection than Lipofectamine (Figure 10B). A similar positive correlation between EGF loading and GFP expression was also observed when CD-PEI2.5k was used in the formulations (Supporting Information Figure S12). Although RAW24.7 cells do have a baseline EGFR expression that varies in response to its activation, Hardbower et al. showed that up to 5 ng/mL EGF did not lead to enhancement in EGFR expression in RAW264.7.64 Since the amount used in our formulations is in the sub-100 pg/mL range, we attribute the increased GFP expression level to enhanced efficacy rather than stimulated EGFR expression. In any case, the tumor-associated macrophages in bladder cancer are typically M2-like (pro-tumorigenic). Since it has been reported that activation of EGFR in such cases mediates inhibition of M2 polarization,65 the uptake of EGF-decorated particles by macrophages may result in abrogating pro-tumorigenic tendencies of the macrophages. Future work testing these particles in vivo will shed more light on this possibility.

Conclusions

Recent work from our lab has shown that a rapid organic solvent extraction-precipitation method for purifying N24-EGF produced material that was capable of targeting EGFR+ bladder tumors tissues in mice (in vitro, ex vivo, and in vivo), dogs (in vitro and ex vivo) and humans (in vitro and ex vivo).28 In an effort to extend this campaign beyond tumor imaging, we sought to develop an ELP carrier system for delivering the nucleic acid cargo. Lecommandoux and co-workers have reported chemically modified ELP grafted with cationic substituents for condensing and delivering pDNA.45,66 The approach reported above is distinct in that the ELP-EGF fusion remains chemically unmodified but is used to engage cationic cyclodextrin:nucleic acid condensates via host:guest interactions with the hydrophobic valine and proline residues of the ELP block. The resulting LENN formulations are shown to be capable of efficiently delivering short (21 bp) nucleic acid sequence cargo such as siRNA as well as large (5 kbp) pDNA constructs using the same components in a LBL assembly process. In addition to the wide range of nucleic acid cargo capacities that this approach affords, it enables the same broad compositional variations offered by LNP approaches without their known liabilities of physical and chemical instability. Importantly, the formulation precursors used in the LENN complexes are biomanufacturable on the scale, chemically stable, display low immunogenicity, and can be tuned to meet the needs of the nucleic acid cargo size. A core design principle of this LENN system is the programmable nanoparticle stability afforded by the cooperativity of multiple host–guest interactions between the ELP prosthetic groups and the CD cavities to provide a tunable balance between the stability need to reach the biological target site and the capacity for programmed disassembly within the target cell to enable efficacious bioactivity (Figure 11). Our previous work using organic extraction-precipitation allows for LPS-free protein purification from E. coli,26,27 while this work shows that LENN formulation and cellular transfection are feasible within the same day starting from the bacterial pellet. In addition to the biomanufacturing advantages provided by this nucleic acid delivery approach, this system is capable of [1] formulation using a LBL approach to provide high adaptability for engaging the desired biological target using blended ELP components to provide the desired functional properties; [2] cargo release kinetics that are governed by the polycationic ponytail on the cyclodextrin and the nature of the ELP; [3] suppression of off-target engagement due to the steric stabilization properties of ELP; [4] obviation of the known liabilities of bioactive lipid formulation due to uncontrolled phase transitions and chemical inactivation by lipidation of the nucleic acid cargo; and [5] siRNA mediated silencing and pDNA expression using a carrier system with low toxicity.

Figure 11 Conceptual diagram of nucleic acid delivery mediated by CD-PLR10:ELP LENN complexes. Step 1: Clathrin-mediated endocytosis of the EGFR-EGF LENN complex; Step 2: Loss of the clathrin cage; Step 3: Sorting and recycling of EGFR; Step 4: Physical exchange of the ELP onto the endosomal membrane surface to expose the polyion core; Steps 5 and 6: Ion exchange between the polycation core and negatively charged phospholipids in the inner endosomal membrane leaflet to release the nucleic acid cargo into the cytosol; Step 7: Loading of siRNA cargo into RISC.

Recent work has shown that different cell types and environments need delivery vehicles with adaptable biochemical, biophysical, and physical properties for the optimal biological outcome. Our aim was to develop an agile peptide-based nucleic acid delivery system to enable rapid evolution of the therapeutic strategy to meet various therapeutic challenges. Our future work will explore the in vivo potential of this system for bladder tumor treatment in an animal model.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00165.Cyclodextrin conjugate synthesis, particle formation, morphological characterization, and biochemical and biological characterization (PDF)

Supplementary Material

bm4c00165_si_001.pdf

Author Contributions

A.A. and S.D. designed and executed the DLS and agarose gel characterization experiments; they also conducted the confocal microscopy and flow cytometry cell culture experiments. A.A. performed the TEM experiments and identified the use of ELP-EGF in LENN formation. K.M.E. performed the AFM experiments. E.Y. assisted with DLS and expressed and purified the ELP constructs using the extraction-precipitation protocol. D.H.T. conceived, designed, directed, and secured funding for the LENN system. A.A., S.D., and D.H.T. wrote the manuscript.

The authors declare no competing financial interest.

Acknowledgments

We thank the Purdue Institute for Cancer Research NCI CCSG grant (CA23168) and the SIRG Fellowship program for financial support of this project. Additional support of this effort by the Flow Cytometry Shared Resource, Chemistry Research Instrumentation Center, Chemical Genomics Facility, and the Purdue Life Science Microscopy Facility at Purdue University is also gratefully acknowledged. The efforts of Marissa Henager and Joshua Mazur in manuscript preparation are also greatly appreciated.

Abbreviations

CD-PEI2.5k, β-cyclodextrin with a single 2.5 kDa polyethylenimine substitution on the 6-hydroxy rim

CD-PLR10, β-cyclodextrin with a single arginine 10-mer substitution on the 6-hydroxy rim

EGFR epidermal growth factor receptor

ELP elastin-like polypeptide

ELP-EGF (N24-EGF) C-terminal epidermal growth factor fusion with a 24-mer ELP repeat, (VPGNG)24

LENN layer-by-layer ELP:nucleic acid nanoparticle

LNP lipid:nucleic acid nanoparticle

NA nucleic acid

N40 40-mer ELP repeat, (VPGNG)40
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