Peptide Amphiphile Nanofibers Research Articles

Microscopically segregated ligand distribution in co-assembled peptide-amphiphile nanofibers

Peptide amphiphiles (PAs) self-assemble into cylindrical nanofibers with applications in protein purification, tissue engineering, and regenerative medicine. For these applications, functionalized PAs are often co-assembled with oppositely charged filler PAs....

Systemic peptide amphiphile nanofiber delivery following subcutaneous injection

Peptide amphiphile (PA) nanofibers have been shown to target and deliver drugs when administered via an intravenous (IV) injection. Subcutaneous administration can broaden the applicability of PA nanofibers in the medical field. The ability of PA nanofibers to be absorbed into systemic circulation after subcutaneous administration was investigated. Four PA molecules with different amino acid sequences were designed to understand the effect of nanofiber cohesion and charge on uptake. Solution small-angle X-ray scattering confirmed nanostructure morphology and provided characteristic lengths for co-assemblies. Circular dichroism and solution wide-angle X-ray scattering confirmed PA secondary structure and molecular order. PAs were co-assembled in a 95 %:5 % molar ratio of unlabeled PA to fluorescently labeled PA. Male and female Sprague Dawley rats were injected in the nape of the neck with PA co-assemblies. In vivo normalized abdominal fluorescence was measured 1–72 h after injection. PA nanofibers with a negative charge and low internal order showed the highest amount of systemic absorption at 1, 6, and 24 h. At 24 h after injection, white blood cell count decreased and glucose was elevated. Glucose began to decrease at 48 h. These data indicate that PA nanofibers can be absorbed into the systemic circulation after subcutaneous injection.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Biomimetic Extracellular Scaffolds by Microfluidic Superstructuring of Nanofibers.

The extracellular matrix is a dynamic framework bearing chemical and morphological cues that support many cellular functions, and artificial analogs with well-defined chemistry are of great interest for biomedical applications. Herein, we describe hierarchical, extracellular-matrix-mimetic microgels, termed "superbundles" (SBs) composed of peptide amphiphile (PA) supramolecular nanofiber networks created using flow-focusing microfluidic devices. We explore the effects of altered flow rate ratio and PA concentration on the ability to create SBs and develop design rules for producing SBs with both cationic and anionic PA nanofibers and gelators. We demonstrate the morphological similarities of SBs to decellularized extracellular matrices and showcase their ability to encapsulate and retain proteinaceous cargos with a wide variety of isoelectric points. Finally, we demonstrate that the novel SB morphology does not affect the well-established biocompatibility of PA gels.

Sustained-release losartan from peptide nanofibers promotes chondrogenesis.

Introduction: The central pathologic feature of osteoarthritis (OA) is the progressive loss of articular cartilage, which has a limited regenerative capacity. The TGF-β1 inhibitor, losartan, can improve cartilage repair by promoting hyaline rather that fibrous cartilage tissue regeneration. However, there are concerns about side effects associated with oral administration and short retention within the joint following intra-articular injections. To facilitate local and sustained intra-articular losartan delivery we have designed an injectable peptide amphiphile (PA) nanofiber that binds losartan. The aims of this study are to characterize the release kinetics of losartan from two different PA nanofiber compositions followed by testing pro-regenerative bioactivity on chondrocytes. Methods: We tested the impact of electrostatic interactions on nanostructure morphology and release kinetics of the negatively charged losartan molecule from either a positively or negatively charged PA nanofiber. Subsequently, cytotoxicity and bioactivity were evaluated in vitro in both normal and an IL-1β-induced OA chondrocyte model using ATDC5. Results: Both nanofiber systems promoted cell proliferation but that the positively-charged nanofibers also significantly increased glycosaminoglycans production. Furthermore, gene expression analysis suggested that losartan-encapsulated nanofibers had significant anti-inflammatory, anti-degenerative, and cartilage regenerative effects by significantly blocking TGF-β1 in this in vitro system. Discussion: The results of this study demonstrated that positively charged losartan sustained-release nanofibers may be a novel and useful treatment for cartilage regeneration and OA by blocking TGF-β1.

MP38-06 SONIC HEDGEHOG SIGNALING IN CORPORA CAVERNOSAL CELLS FROM PROSTATECTOMY, DIABETIC, HYPERTENSION AND PEYRONIE’S PATIENTS WITH ERECTILE DYSFUNCTION

You have accessJournal of UrologyCME1 May 2022MP38-06 SONIC HEDGEHOG SIGNALING IN CORPORA CAVERNOSAL CELLS FROM PROSTATECTOMY, DIABETIC, HYPERTENSION AND PEYRONIE’S PATIENTS WITH ERECTILE DYSFUNCTION Sarah Martin, Timothy Searl, Samuel Ohlander, Kevin McVary, and Carol Podlasek Sarah MartinSarah Martin More articles by this author , Timothy SearlTimothy Searl More articles by this author , Samuel OhlanderSamuel Ohlander More articles by this author , Kevin McVaryKevin McVary More articles by this author , and Carol PodlasekCarol Podlasek More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002592.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Erectile dysfunction (ED) treatments are minimally effective in prostatectomy and diabetic patients due to injury to the cavernous nerve. With denervation the critical smooth muscle undergoes apoptosis and the penis becomes fibrotic, thus altering corpora cavernosal architecture. In order to devise novel ED therapies, prevention of corpora cavernosal remodeling is critical. Sonic hedgehog (SHH) treatment by peptide amphiphile (PA) nanofiber hydrogel, suppresses smooth muscle apoptosis and improves erectile function in a rat CN injury ED model. We examine if human corpora cavernosal smooth muscle responds in a similar manner to SHH treatment.In this study we: 1.) Examine if human corpora cavernosal smooth muscle cells from primary culture of ED patient tissues, respond to Sonic hedgehog (SHH) treatment by increasing growth rate, as in our in vivo animal model. 2.) Determine if human corpora cavernosal cells from patients with differing underlying mechanisms of ED development (prostatectomy, diabetes, hypertension, cardiovascular disease), respond similarly to SHH treatment. 3.) Identify if organ culture conditions (glucose) affect growth of corpora cavernosal smooth muscle and the response to SHH signaling. 4.) Examine the vascular component of ED (hypertension and cardiovascular disease). METHODS: Human corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie’s (control) patients (n=40) that were under going prosthesis implant to treat ED. Primary cultures (n=17) were established, and corpora cavernosal cells from passage 3-4 were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells using a hemocytometer at 3-4 days. The concentration of SHH protein for maximal growth was examined, in addition to a lipid modified more active SHH peptide. RESULTS: SHH treatment increased smooth muscle growth in human corpora cavernosal cells from prostatectomy, diabetic, and Peyronie’s patients in a similar manner (43-53%). SHH inhibition decreased smooth muscle growth (24-32%). There was no difference in growth using 25ug and 10ug SHH peptide. A more active (150X) SHH peptide further enhanced growth (20%). SHH protein increased growth more in diabetic smooth muscle under high glucose conditions. SHH inhibition was less effective under high glucose conditions. SHH treatment increased growth less in cells from hypertension and cardiovascular disease patients. CONCLUSIONS: Corpora cavernosa smooth muscle from prostatectomy, diabetic, and Peyronie’s patients increased in response to SHH treatment in a similar manner, suggesting that SHH treatment would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple origins. There is a threshold concentration of SHH protein above which smooth muscle growth is enhanced. Lipid modified SHH engendered a more robust growth response. High glucose conditions that may be present in under controlled diabetic patients would not detract from SHH usefulness as a protectant for corpora cavernosal morphology. Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation to ED patients. Source of Funding: NIH/NIDDK DK101536 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e620 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sarah Martin More articles by this author Timothy Searl More articles by this author Samuel Ohlander More articles by this author Kevin McVary More articles by this author Carol Podlasek More articles by this author Expand All Advertisement PDF DownloadLoading ...

Sonic hedgehog signaling in corpora cavernosal cells from prostatectomy, diabetic, hypertension and Peyronie's patients with erectile dysfunction

ABSTRACT Introduction Erectile dysfunction (ED) treatments are minimally effective in prostatectomy and diabetic patients due to injury to the cavernous nerve. With denervation the critical smooth muscle undergoes apoptosis and the penis becomes fibrotic, thus altering corpora cavernosal architecture. In order to devise novel ED therapies, prevention of corpora cavernosal remodeling is critical. Sonic hedgehog (SHH) treatment by peptide amphiphile (PA) nanofiber hydrogel, suppresses smooth muscle apoptosis and improves erectile function in a rat CN injury ED model. We examine if human corpora cavernosal smooth muscle responds in a similar manner to SHH treatment. Objective In this study we: 1.) Examine if human corpora cavernosal smooth muscle cells from primary culture of ED patient tissues, respond to Sonic hedgehog (SHH) treatment by increasing growth rate, as in our in vivo animal model. 2.) Determine if human corpora cavernosal cells from patients with differing underlying mechanisms of ED development (prostatectomy, diabetes, hypertension, cardiovascular disease), respond similarly to SHH treatment. 3.) Identify if organ culture conditions (glucose) affect growth of corpora cavernosal smooth muscle and the response to SHH signaling. 4.) Examine the vascular component of ED (hypertension and cardiovascular disease). Methods Human corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie's (control) patients (n=40) that were under going prosthesis implant to treat ED. Primary cultures (n=17) were established, and corpora cavernosal cells from passage 3-4 were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells using a hemocytometer at 3-4 days. The concentration of SHH protein for maximal growth was examined, in addition to a lipid modified more active SHH peptide. Results SHH treatment increased smooth muscle growth in human corpora cavernosal cells from prostatectomy, diabetic, and Peyronie's patients in a similar manner (43-53%). SHH inhibition decreased smooth muscle growth (24-32%). There was no difference in growth using 25ug and 10ug SHH peptide. A more active (150X) SHH peptide further enhanced growth (20%). SHH protein increased growth more in diabetic smooth muscle under high glucose conditions. SHH inhibition was less effective under high glucose conditions. SHH treatment increased growth less in cells from hypertension and cardiovascular disease patients. Conclusion Corpora cavernosa smooth muscle from prostatectomy, diabetic, and Peyronie's patients increased in response to SHH treatment in a similar manner, suggesting that SHH treatment would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple origins. There is a threshold concentration of SHH protein above which smooth muscle growth is enhanced. Lipid modified SHH engendered a more robust growth response. High glucose conditions that may be present in under controlled diabetic patients would not detract from SHH usefulness as a protectant for corpora cavernosal morphology. Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation to ED patients. Disclosure Work supported by industry: no.

Sonic Hedgehog Signaling in Corpora Cavernosal Cells from Prostatectomy, Diabetic, Hypertension and Peyronie's Patients with Erectile Dysfunction

ABSTRACT Introduction Erectile dysfunction (ED) treatments are minimally effective in prostatectomy and diabetic patients due to injury to the cavernous nerve. With denervation the critical smooth muscle undergoes apoptosis and the penis becomes fibrotic, thus altering corpora cavernosal architecture. In order to devise novel ED therapies, prevention of corpora cavernosal remodeling is critical. Sonic hedgehog (SHH) treatment by peptide amphiphile (PA) nanofiber hydrogel, suppresses smooth muscle apoptosis and improves erectile function in a rat CN injury ED model. We examine if human corpora cavernosal smooth muscle responds in a similar manner to SHH treatment. Objective In this study we: 1.) Examine if human corpora cavernosal smooth muscle cells from primary culture of ED patient tissues, respond to Sonic hedgehog (SHH) treatment by increasing growth rate, as in our in vivo animal model. 2.) Determine if human corpora cavernosal cells from patients with differing underlying mechanisms of ED development (prostatectomy, diabetes, hypertension, cardiovascular disease), respond similarly to SHH treatment. 3.) Identify if organ culture conditions (glucose) affect growth of corpora cavernosal smooth muscle and the response to SHH signaling. 4.) Examine the vascular component of ED (hypertension and cardiovascular disease). Methods Human corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie's (control) patients (n=40) that were under going prosthesis implant to treat ED. Primary cultures (n=17) were established, and corpora cavernosal cells from passage 3-4 were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells using a hemocytometer at 3-4 days. The concentration of SHH protein for maximal growth was examined, in addition to a lipid modified more active SHH peptide. Results SHH treatment increased smooth muscle growth in human corpora cavernosal cells from prostatectomy, diabetic, and Peyronie's patients in a similar manner (43-53%). SHH inhibition decreased smooth muscle growth (24-32%). There was no difference in growth using 25ug and 10ug SHH peptide. A more active (150X) SHH peptide further enhanced growth (20%). SHH protein increased growth more in diabetic smooth muscle under high glucose conditions. SHH inhibition was less effective under high glucose conditions. SHH treatment increased growth less in cells from hypertension and cardiovascular disease patients. Conclusions Corpora cavernosa smooth muscle from prostatectomy, diabetic, and Peyronie's patients increased in response to SHH treatment in a similar manner, suggesting that SHH treatment would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple origins. There is a threshold concentration of SHH protein above which smooth muscle growth is enhanced. Lipid modified SHH engendered a more robust growth response. High glucose conditions that may be present in under controlled diabetic patients would not detract from SHH usefulness as a protectant for corpora cavernosal morphology. Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation to ED patients. Disclosure No

Acid-Base Equilibrium and Dielectric Environment Regulate Charge in Supramolecular Nanofibers.

Peptide amphiphiles are a class of molecules that can self-assemble into a variety of supramolecular structures, including high-aspect-ratio nanofibers. It is challenging to model and predict the charges in these supramolecular nanofibers because the ionization state of the peptides are not fixed but liable to change due to the acid-base equilibrium that is coupled to the structural organization of the peptide amphiphile molecules. Here, we have developed a theoretical model to describe and predict the amount of charge found on self-assembled peptide amphiphiles as a function of pH and ion concentration. In particular, we computed the amount of charge of peptide amphiphiles nanofibers with the sequence C 16 − V 2 A 2 E 2. In our theoretical formulation, we consider charge regulation of the carboxylic acid groups, which involves the acid-base chemical equilibrium of the glutamic acid residues and the possibility of ion condensation. The charge regulation is coupled with the local dielectric environment by allowing for a varying dielectric constant that also includes a position-dependent electrostatic solvation energy for the charged species. We find that the charges on the glutamic acid residues of the peptide amphiphile nanofiber are much lower than the same functional group in aqueous solution. There is a strong coupling between the charging via the acid-base equilibrium and the local dielectric environment. Our model predicts a much lower degree of deprotonation for a position-dependent relative dielectric constant compared to a constant dielectric background. Furthermore, the shape and size of the electrostatic potential as well as the counterion distribution are quantitatively and qualitatively different. These results indicate that an accurate model of peptide amphiphile self-assembly must take into account charge regulation of acidic groups through acid–base equilibria and ion condensation, as well as coupling to the local dielectric environment.

Peptide Amphiphile Hydrogels Based on Homoternary Cucurbit[8]uril Host-Guest Complexes.

Supramolecular hydrogels based on peptide amphiphiles (PAs) are promising materials for tissue engineering and model extracellular matrixes for biological studies. While PA hydrogels are conventionally formed via electrostatic screening, new hydrogelation mechanisms might help to improve the design and functionality of these materials. Here, we present a host-guest-mediated PA hydrogelation method that relies on the formation of a host-guest homoternary complex with cucurbit[8]uril (CB[8]) and aromatic amino-acid-bearing PA nanofibers. As a result of the host-guest cross-linking between PA nanofibers, hierarchical morphologies and increased stiffness were found when host-guest-mediated PA hydrogels were compared to their ion-based equivalents. Additionally, both families of hydrogels exhibited similar biocompatibilities. These results demonstrate that CB[8]-mediated hydrogelation can be used as an alternative cross-linking method to upgrade the design of PA materials and extend their biomedical applications.

Hydrogen Bonding Stiffens Peptide Amphiphile Supramolecular Filaments by Aza-Glycine Residues

Peptide amphiphiles (PAs) are a class of molecules comprised of short amino acid sequences conjugated to hydrophobic moieties that may exhibit self-assembly in water into supramolecular structures. We investigate here how mechanical properties of hydrogels formed by PA supramolecular nanofibers are affected by hydrogen bond densities within their internal structure by substituting glycine for aza-glycine (azaG) residues. We found that increasing the number of PA molecules that contain azaG up to 5 mol% in PA supramolecular nanofibers increases their persistence length fivefold and decreases their diffusion coefficients as measured by fluorescence recovery after photobleaching. When these PAs are used to create hydrogels, their bulk storage modulus (G′) was found to increase as azaG PA content in the supramolecular assemblies increases up to a value of 10 mol% and beyond this value a decrease was observed, likely due to diminished levels of nanofiber entanglement in the hydrogels as a direct result of increased supramolecular rigidity. Interestingly, we found that the bioactivity of the scaffolds toward dopaminergic neurons derived from induced pluripotent stem cells can be enhanced directly by persistence length independently of storage modulus. We hypothesize that this is due to interactions between the cells and the extracellular environment across different size scales: from filopodia adhering to individual nanofiber bundles to cell adhesion sites that interact with the hydrogel as a bulk substrate. Fine tuning of hydrogen bond density in self-assembling peptide biomaterials such as PAs provides an approach to control nanoscale stiffness as part of an overall strategy to optimize bioactivity in these supramolecular systems. supramolecular biomaterials. Statement of significanceHydrogen bonding is an important driving force for the self-assembly of peptides in both biological and artificial systems. Here, we increase the amount of hydrogen bonding within self-assembled peptide amphiphile (PA) nanofibers by substituting glycine for an aza-glycine (azaG). We show that increasing the molar concentration of azaG increases the internal order of individual nanofibers and increases their persistence length. We also show that these changes are sufficient to increase survival and tyrosine hydroxylase expression in induced pluripotent stem cell-derived dopaminergic neurons cultured in 3D gels made of these materials. Our strategy of tuning the number of hydrogen bonds in a supramolecular assembly provides mechanical customization for 3D cell culture and tissue engineering.

Peptide amphiphile nanofiber hydrogel delivery of Sonic hedgehog protein to the penis and cavernous nerve suppresses intrinsic and extrinsic apoptotic signaling mechanisms, which are an underlying cause of erectile dysfunction

Erectile dysfunction (ED) is a common and debilitating condition with high impact on quality of life. An underlying cause of ED is apoptosis of penile smooth muscle, which occurs with cavernous nerve injury, in prostatectomy, diabetic and aging patients. We are developing peptide amphiphile (PA) nanofiber hydrogels as an in vivo delivery vehicle for Sonic hedgehog protein to the penis and cavernous nerve to prevent the apoptotic response. We examine two important aspects required for clinical application of the biomaterials: if SHH PA suppresses intrinsic (caspase 9) and extrinsic (caspase 8) apoptotic mechanisms, and if suppressing one apoptotic mechanism forces apoptosis to occur via a different mechanism. We show that SHH PA suppresses both caspase 9 and 8 apoptotic mechanisms, and suppressing caspase 9 did not shift signaling to caspase 8. SHH PA has significant clinical potential as a preventative ED therapy, by management of intrinsic and extrinsic apoptotic mechanisms.

Self-Assembled Peptide Amphiphile Nanofibers for Controlled Therapeutic Delivery to the Atherosclerotic Niche.

Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers were developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs released LXR in response to physiological levels of ROS and reducing agents and could be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers targeted plaque within an atherosclerotic mouse model in vivo and activated ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.

Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice.

Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non-specific biodistribution. Self-assembled peptide amphiphile (PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off-target toxicity. Here, PA nanofibers that target the angiotensin I-converting enzyme and the receptor for advanced glycation end-products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE-targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia-induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off-target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE-targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.

Development of novel nanofibers targeted to smoke-injured lungs

Smoke inhalation injury is associated with significant mortality and current therapies remain supportive. The purpose of our study was to identify proteins upregulated in the lung after smoke inhalation injury and develop peptide amphiphile nanofibers that target these proteins. We hypothesize that nanofibers targeted to angiotensin-converting enzyme or receptor for advanced glycation end products will localize to smoke-injured lungs. MethodsFive targeting sequences were incorporated into peptide amphiphile monomers methodically to optimize nanofiber formation. Nanofiber formation was assessed by conventional transmission electron microscopy. Rats received 8 min of wood smoke. Levels of angiotensin-converting enzyme and receptor for advanced glycation end products were evaluated by immunofluorescence. Rats received the targeted nanofiber 23 h after injury via tail vein injection. Nanofiber localization was determined by fluorescence quantification. ResultsPeptide amphiphile purity (>95%) and nanofiber formation were confirmed. Target proteins were increased in smoke inhalation versus sham (p < 0.001). After smoke inhalation and injection of targeted nanofibers, we found a 10-fold increase in angiotensin-converting enzyme-targeted nanofiber localization to lung (p < 0.001) versus sham with minimal localization of non-targeted nanofiber (p < 0.001). ConclusionsWe synthesized, characterized, and evaluated systemically delivered targeted nanofibers that localized to the site of smoke inhalation injury in vivo. Angiotensin-converting enzyme-targeted nanofibers serve as the foundation for developing a novel nanotherapeutic that treats smoke inhalation lung injury.

Hierarchical Superstructures: Superstructured Biomaterials Formed by Exchange Dynamics and Host–Guest Interactions in Supramolecular Polymers (Adv. Sci. 8/2021)

Dynamic and reversible assembly of molecules is ubiquitous in the hierarchical superstructures of living systems and plays a key role in cellular functions. In article number 2004042 by Samuel I. Stupp and co-workers, host -guest interactions between b-cyclodextrin and adamantane-functionalized peptide amphiphiles led to analogous superstructure formation among nanofibers. The cover displays this hierarchical superstructure formation of fluorescently labelled peptide amphiphile nanofibers captured by confocal microscopy.

A Chemotactic Functional Scaffold with VEGF-Releasing Peptide Amphiphiles Facilitates Bone Regeneration by BMP-2 in a Large-Scale Rodent Cranial Defect Model.

Current common techniques for repairing calvarial defects by autologous bone grafting and alloplastic implants have significant limitations. In this study, the authors investigated a novel alternative approach to bone repair based on peptide amphiphile nanofiber gels that are engineered to control the release of vascular endothelial growth factor (VEGF) to recruit circulating stem cells to a site of bone regeneration and facilitate bone healing by bone morphogenetic protein-2 (BMP-2). VEGF release kinetics from peptide amphiphile gels were evaluated. Chemotactic functional scaffolds were fabricated by combining collagen sponges with peptide amphiphile gels containing VEGF. The in vitro and in vivo chemotactic activities of the scaffolds were evaluated by measuring mesenchymal stem cell migration, and angiogenic capability of the scaffolds was also evaluated. Large-scale rodent cranial bone defects were created to evaluate bone regeneration after implanting the scaffolds and other control materials. VEGF was released from peptide amphiphile in a controlled-release manner. In vitro migration of mesenchymal stem cells was significantly greater when exposed to chemotactic functional scaffolds compared to control scaffolds. In vivo chemotaxis was evidenced by migration of tracer-labeled mesenchymal stem cells to the chemotactic functional scaffolds. Chemotactic functional scaffolds showed significantly increased angiogenesis in vivo. Successful bone regeneration was noted in the defects treated with chemotactic functional scaffolds and BMP-2. The authors' observations suggest that this bioengineered construct successfully acts as a chemoattractant for circulating mesenchymal stem cells because of controlled release of VEGF from the peptide amphiphile gels. The chemotactic functional scaffolds may play a role in the future design of clinically relevant bone graft substitutes for large-scale bone defects.

Multivalent Clustering of Adhesion Ligands in Nanofiber-Nanoparticle Composites

Because the positioning and clustering of biomolecules within the extracellular matrix dictates cell behaviors, the engineering of biomaterials incorporating bioactive epitopes with spatial organization tunable at the nanoscale is of primary importance. Here we used a highly modular composite approach combining peptide amphiphile (PA) nanofibers and silica nanoparticles, which are both easily functionalized with one or several bioactive signals. We show that the surface of silica nanoparticles allows the clustering of RGDS bioactive signals leading to improved adhesion and spreading of fibroblast cells on composite hydrogels at an epitope concentration much lower than in PA-only based matrices. Most importantly, by combining the two integrin-binding sequences RGDS and PHSRN on nanoparticle surfaces, we improved cell adhesion on the PA nanofiber/particle composite hydrogels, which is attributed to synergistic interactions known to be effective only for peptide intermolecular distance of ca. 5 nm. Such composites with soft and hard nanostructures offer a strategy for the design of advanced scaffolds to display multiple signals and control cell behavior.

Epitope Peptide Amphiphile-Based Nanofiber as an Effective Vaccine for Viral Infectious Diseases.

Peptide-based vaccines are relatively safe but have weak immune responses even with an adjuvant. In order to overcome the limitations of peptide-based vaccines, we developed peptide amphiphile (PA)-based nanofibers to enhance the immune responses for preventing enterovirus 71 (EV71) infectious disease (i.e., Hand, Foot, and Mouth Disease). PAs are peptides conjugated with fatty acid alkyl chain and able to self-assemble into various structures including high-aspectratio nanofibers. We designed PAs by coupling EV71 virus particle 1 (VP1) epitope peptides and spacer-crosslinker to the N-terminal of long-chain fatty acids (VP1-PA). PAs then self-assembled into nanofibers at physiological pH (pH 7.4). PA nanofibers were characterized by atomic force microscopy (AFM). For the immunization studies, C57BL/6 mice were injected intraperitoneally (i.p.) with recombinant VP1 with adjuvant (alum), VP1 epitope peptide with or without adjuvant, VP1-PA nanofibers with or without adjuvant, and PBS. To assess the immunogenecity of the VP1-PA nanofibers on serum samples from the immunized mice was analyzed by Western blot for the evaluation of VP1-specific IgG. The PA group showed a higher immune response than the peptide group. We expect that self-assembling VP1-PA based nanofibers as an immune stimulator could enhance immune responses effectively against EV71 infection and overcome the limitations of peptide-based vaccine.

Unravelling the Enzymatic Degradation Mechanism of Supramolecular Peptide Nanofibers and Its Correlation with Their Internal Viscosity.

Enzyme-responsive supramolecular peptide biomaterials have attracted growing interest for disease diagnostics and treatments. However, it remains unclear whether enzymes target the peptide assemblies or dissociated peptide monomers. To gain further insight into the degradation mechanism of supramolecular peptide amphiphile (PA) nanofibers, cathepsin B with both exopeptidase and endopeptidase activities was exploited here for degradation studies. Hydrolysis was found to occur directly on the PA nanofibers as only surface amino acid residues were cleaved. The number of cleaved residues and the degradation efficiency was observed to be negatively correlated with the internal viscosity of the PA nanofibers, quantified to be between 200-800 cP (liquid phase) using fluorescence lifetime imaging microscopy combined with an environmentally sensitive molecular rotor, BODIPY-C10. These findings enhance our understanding on the enzymatic degradation of supramolecular PA nanofibers and have important implications for the development of PA probes for the real-time monitoring of disease-related enzymes.