R-3 Repository :: Browsing by Author "Hartgerink, Jeffrey D."

Browsing by Author "Hartgerink, Jeffrey D."

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A Journey through the Arabidopsis thaliana Genome: Discovering the Origins of Novel Triterpene Metabolites
(2014-08-27) Castillo-Rivera, Dorianne A; Matsuda, Seiichi P. T.; Hartgerink, Jeffrey D.; Tapia, Richard A.
Plants produce a large variety of natural products, including over 20,000 different triterpenoids. Triterpenoid functions range from roles as membrane sterols and hormones in primary metabolism, to defense compounds in secondary metabolism. Beyond these roles in the plant, triterpenoids have substantial human value as flavors, fragrances, and medicines. This thesis explores the enzymatic formation of triterpenoids through cationic cyclization of a linear precursor oxidosqualene and further metabolism by radical oxidation. Thirteen different oxidosqualene cyclases (OSCs) are encoded by the model plant Arabidopsis thaliana and collectively produce a plethora of triterpene skeletons. This structural diversity of triterpenes was investigated through a comprehensive analysis of the A. thaliana PEN6 product profile. This product profile contained 33 compounds that were found by combining genome mining, heterologous expression in yeast and HSQC analysis. Some of these compounds were novel to Arabidopsis: isoursenol, (13R,14Z,17E)-malabarica-14,17,21-trien-3β-ol, nematocyphol, (20R,S) dammarenediols, Δ8(26)-seco-β-amyrin, and 9αH-Δ8(26)-polypodatetraenol. After the cyclization, triterpenes can be modified by a number of enzymes, including cytochrome P450 monooxygenases (CYP450s). Candidate CYP450s for a given triterpene were identified by gene cluster analysis combined with microarray databases. Heterologous expression of the OSC, ATR2 and CYP450 together with GC-MS and NMR techniques allowed the elucidation of metabolic pathways and structures of a variety of oxygenated triterpenes. These experimental techniques led to the identification of new oxidized metabolites produced by the co-expression of Arabidopsis clusters: THAS1 with CYP708A2 and CYP705A5, PEN1 with CYP705A2 and MRN1 with CYP71A16. The thalianol cluster gave rise to several unexpected side chain and ring oxidized metabolites. This thesis also describes the side chain cleavage of arabidiol by CYP705A1 to give a C19 methyl ketone, and the hydroxylation of an allylic methyl of marneral/marnerol to 23-hydroxymarneral/23-hydroxymarnerol by CYP71A16. This work sheds light on the metabolic fate of some Arabidopsis triterpenes. When applied more generally, this strategy may begin to fill a large knowledge gap in metabolomics and functional genomics.
A Nanostructured Synthetic Collagen Mimic for Hemostasis
(American Chemical Society, 2014) Kumar, Vivek A.; Taylor, Nichole L.; Jalan, Abhishek A.; Hwang, Lyahn K.; Wang, Benjamin K.; Hartgerink, Jeffrey D.
Collagen is a major component of the extracellular matrix and plays a wide variety of important roles in blood clotting, healing, and tissue remodeling. Natural, animal derived, collagen is used in many clinical applications but concerns exist with respect to its role in inflammation, batch-to-batch variability, and possible disease transfection. Therefore, development of synthetic nanomaterials that can mimic the nanostructure and properties of natural collagen has been a heavily pursued goal in biomaterials. Previously, we reported on the design and multihierarchial self-assembly of a 36 amino acid collagen mimetic peptide (KOD) that forms nanofibrous triple helices that entangle to form a hydrogel. In this report, we utilize this nanofiber forming collagen mimetic peptide as a synthetic biomimetic matrix useful in thrombosis. We demonstrate that nanofibrous KOD synthetic collagen matrices adhere platelets, activate them (indicated by soluble P-selectin secretion), and clot plasma and blood similar to animal derived collagen and control surfaces. In addition to the thrombotic potential, THP-1 monocytes incubated with our KOD collagen mimetic showed minimal proinflammatory cytokine (TNF-α or IL-1β) production. Together, the data presented demonstrates the potential of a novel synthetic collagen mimetic as a hemostat.
A wireless millimetric magnetoelectric implant for the endovascular stimulation of peripheral nerves
(Springer Nature, 2022) Chen, Joshua C.; Kan, Peter; Yu, Zhanghao; Alrashdan, Fatima; Garcia, Roberto; Singer, Amanda; Lai, C.S. Edwin; Avants, Ben; Crosby, Scott; Li, Zhongxi; Wang, Boshuo; Felicella, Michelle M.; Robledo, Ariadna; Peterchev, Angel V.; Goetz, Stefan M.; Hartgerink, Jeffrey D.; Sheth, Sunil A.; Yang, Kaiyuan; Robinson, Jacob T.; Applied Physics Program
Implantable bioelectronic devices for the simulation of peripheral nerves could be used to treat disorders that are resistant to traditional pharmacological therapies. However, for many nerve targets, this requires invasive surgeries and the implantation of bulky devices (about a few centimetres in at least one dimension). Here we report the design and in vivo proof-of-concept testing of an endovascular wireless and battery-free millimetric implant for the stimulation of specific peripheral nerves that are difficult to reach via traditional surgeries. The device can be delivered through a percutaneous catheter and leverages magnetoelectric materials to receive data and power through tissue via a digitally programmable 1 mm × 0.8 mm system-on-a-chip. Implantation of the device directly on top of the sciatic nerve in rats and near a femoral artery in pigs (with a stimulation lead introduced into a blood vessel through a catheter) allowed for wireless stimulation of the animals’ sciatic and femoral nerves. Minimally invasive magnetoelectric implants may allow for the stimulation of nerves without the need for open surgery or the implantation of battery-powered pulse generators.
Charge Pairs and Mutations in Collagen Mimetic Peptides
(2017-04-10) Clements, Katherine Ann; Hartgerink, Jeffrey D.
This thesis will present insights into natural type I collagen based upon studies with collagen mimetic peptides. Natural collagen is a fibrous protein with challenging properties, such as solubility, which makes it difficult to study through analytical methods such as nuclear magnetic resonance spectroscopy. Collagen mimetic peptides are a flexible and controlled model system that imitates natural type I collagen structure, a triple helix, on a smaller scale, from over 1000 amino acids to around 30 amino acids. Chapter one will explore the development of collagen mimetic peptides and review the current design principle to form composition and register specific triple helices. Chapter two will illustrate the power of charge pairing in the triple helix with the creation of a triple helix with an extended offset through axial charge pairing. This is the first triple helix with an extended offset, proving that the offset triple helix is possible, even when it is not incorporated into a fiber. Chapters three and four will model mutations from Osteogenesis Imperfecta, a type I collagen disease, in collagen mimetic peptides. Osteogenesis Imperfecta results from mutations of the requisite glycines in the Xaa-Yaa-Gly repeat of collagen. Mutations in both the A and B chains of an AAB triple helix will be created, and their impact on the composition, stability, and structure of the triple helix will be examined through circular dichroism and nuclear magnetic resonance experiments. The results of those experiments will be used to create molecular models of the mutated triple helices. This series of studies gives insight into the forces in the triple helix: the destabilizing force of a mutation, the stabilizing force of hydrogen bonds, and the stabilizing, yet constricting, force of axial charge pairs. In these model systems, the mutation was able to direct the composition and register of the triple helix, where the triple helix with the lowest number of mutations would fold, even if it is not the most stabilized via charge pairing.
Controlled Angiogenesis in Peptide Nanofiber Composite Hydrogels
(American Chemical Society, 2015) Wickremasinghe, Navindee C.; Kumar, Vivek A.; Shi, Siyu; Hartgerink, Jeffrey D.
Multidomain peptide (MDP) nanofibers create scaffolds that can present bioactive cues to promote biological responses. Orthogonal self-assembly of MDPs and growth-factor-loaded liposomes generate supramolecular composite hydrogels. These composites can act as delivery vehicles with time-controlled release. Here we examine the controlled release of placental growth factor-1 (PlGF-1) for its ability to induce angiogenic responses. PlGF-1 was loaded either in MDP matrices or within liposomes bound inside MDP matrices. Scaffolds showed expected rapid infiltration of macrophages. When released through liposomes incorporated in MDP gels (MDP(Lipo)), PlGF-1 modulates HUVEC VEGF receptor activation in vitro and robust vessel formation in vivo. These loaded MDP(Lipo) hydrogels induce a high level of growth-factor-mediated neovascular maturity. MDP(Lipo) hydrogels offer a biocompatible and injectable platform to tailor drug delivery and treat ischemic tissue diseases.
Covalent Capture of Aligned Self-Assembling Nanofibers
(American Chemical Society, 2017) Li, I-Che; Hartgerink, Jeffrey D.
A great deal of effort has been invested in the design and characterization of systems which spontaneously assemble into nanofibers. These systems are interesting for their fundamental supramolecular chemistry and have also been shown to be promising materials, particularly for biomedical applications. Multidomain peptides are one such assembler, and in previous work we have demonstrated the reversibility of their assembly under mild and easily controlled conditions, along with their utility for time-controlled drug delivery, protein delivery, cell encapsulation, and cell delivery applications. Additionally, their highly compliant criteria for sequence selection allows them to be modified to incorporate protease susceptibility and biological-recognition motifs for cell adhesion and angiogenesis. However, control of their assembly has been limited to the formation of disorganized nanofibers. In this work, we expand our ability to manipulate multidomain-peptide assembly into parallel-aligned fiber bundles. Albeit this alignment is achieved by the shearing forces of syringe delivery, it is also dependent on the amino acid sequence of the multidomain peptide. The incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine) allows the self-assembled nanofibers to form an anisotropic hydrogel string under modest shear stress. The hydrogel string shows remarkable birefringence, and highly aligned nanofibers are visible in scanning electronic microscopy. Furthermore, the covalent linkage induced by DOPA oxidation allows covalent capture of the aligned nanofiber bundles, enhancing their birefringence and structural integrity.
Design and Structural Characterization of Self-Assembling Triple Helical Heterotrimers
(2013-06-05) Fallas Valverde, Jorge; Hartgerink, Jeffrey D.; Wolynes, Peter G.; Tao, Yizhi Jane
Design of self-assembling ABC-type collagen triple helical heterotrimers is challenging due to the number of competing species that can be formed in ternary mixture of peptides with a high propensity to fold into triple helices and the fact that well understood rules for pair-wise amino acid stabilization of the canonical collagen triple helix have remained elusive. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population towards a desired target but the presence of competing states in mixtures has remained an outstanding problem. In this work we use high-resolution structural biology techniques to do a detailed study of stabilizing pair-wise interactions between positively and negatively charged amino acids in triple helices. Two types of contacts with distinct sequence requirements depending on the relative stagger of the interacting chains are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. Using this knowledge we developed distinct design strategies to improve the specificity of our designed systems towards the desired ABC heterotrimeric target state. We validate our strategies through the synthesis and characterization of the designed sequences and show that they self-assemble into a highly stable ABC triple helices with control over composition in the case of the rational approach and with control over both composition and register in the case of the computational approach.
Design of Heterotrimeric Collagen Triple Helices
(2014-04-24) Jalan, Abhishek; Hartgerink, Jeffrey D.; Wolynes, Peter G.; Tao, Yizhi Jane
Select loci in native collagen display clusters of contiguous amino acids that recognize a diverse array of extracellular matrix (ECM) and blood serum proteins critical for homeostasis and hemostasis. The mechanism of collagen binding to these proteins has primarily been elucidated using short peptides, called collagen mimetic peptides (CMPs), that independently fold into the so called homotrimeric collagen triple helices, where all three peptide chains have identical amino acid sequence. However, the homotrimer binding mechanism cannot be extrapolated to explain protein binding in AAB and ABC-type heterotrimeric collagens that contain either two or three unique polypeptide chains without significant speculation. Given the requirement of a one amino acid offset between the peptide chains in a collagen triple helix, a mixture of two or three unique peptides can self-assemble into 8 and 27 competing triple helices, respectively. Heterotrimeric CMPs have remained synthetically inaccessible due to the challenge associated with introducing bias in this ensemble of competing states. Previously, Hartgerink lab employed axial Lys – Asp / Glu salt-bridges to successfully self-assemble an ABC heterotrimer. Here, we extend this paradigm to successfully demonstrate the design of a proof-of-principle AAB heterotrimer. Four AAB heterotrimers, each carrying unpaired Lys, Asp or Glu and a combination of Lys – Asp or Lys – Glu axial salt-bridges, were designed. Of these, only the heterotrimer containing unpaired Glu and a combination of Lys – Asp as well as Lys – Glu salt-bridges successfully self-assembled into an AAB heterotrimer. Next, a general methodology to self-assemble AAB heterotrimers containing the α2β1 and α1β1-integrin recognition sequences from collagen I and IV, respectively, and the matrix metalloproteinase-1 cleavage sequence from collagen I was developed. The protein recognition sequences were included as guests in a host peptide sequence containing a network of salt-bridges that bias the ensemble of competing triple helices to the desired AAB heterotrimer. Successful self-assembly of heterotrimers across multiple guest sequences was observed, which demonstrates the wide applicability of the host sequence design. In future, binding of these heterotrimers to the ECM and blood serum proteins has the potential to unravel the mechanism of disease evolution across multiple disease settings. We also extended the salt-bridge based design paradigm to synthesize a new class of CMP constructs. Hydroxyproline-free collagen triple helices are lucrative for expression in bacterial systems. Using a combination of Lys – Asp and Lys – Glu salt-bridges, a hydroxyproline-free ABC collagen heterotrimer was successfully designed. Remarkably, this ABC heterotrimer was stable despite one the peptides containing no proline or hydroxyproline, a requirement previously thought to be critical for stability. Additionally, an ABC heterotrimer containing a non-canonical four residue offset between the peptide chains was designed. In this heterotrimer, the non-covalent interactions at the termini are unsatisfied which renders them “sticky” to further assembly. This design lays the groundwork to create longer and therefore, stickier offsets to facilitate self-assembly of collagen-mimetic nanofibers.
Design of Multidomain Peptides and Collagen Mimetic Peptides for Biological Applications
(2018-04-05) Li, I-Che; Hartgerink, Jeffrey D.
Supramolecular chemistry plays an important role in designing self-assembling peptides. The goal of this work is to design, synthesize, and customize multidomain peptides (MDPs) and collagen mimetic peptides (CMPs) for biological applications. In the first part of this work, we modify the MDP nanofibers to achieve controlled drug release and macroscopic anisotropy. In the second part, we explored residue substitutions in CMPs to capture helical conformations and to target natural collagens. As a type of peptide hydrogel, MDPs respond to external shearing forces and have reversible self-assembly under mild conditions. Controlled drug release is accomplished by modifying the hydrophobic interior of the MDP to construct hollow fibers for the encapsulation of anti-cancer drugs, antibiotics, or nonsteroidal anti-inflammatory drugs. Macroscopic anisotropy was achieved by modifying the hydrophilic exterior of the MDP to achieve organized self-assembly into parallel aligned fiber bundles. With the help of shearing forces of syringe extrusion and the incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine), the self-assembled nanofibers form an anisotropic hydrogel string under modest shear stress. The anisotropic texture was further crosslinked by oxidative polymerization of DOPA residues and pushed to a new level for tissue regeneration. Collagen forms trimeric helical structures with various thermal stability, which is highly related to the residue substitutions. In our CMP design for collagen covalent capture, lysine and aspartate were employed in a trimeric helical structure to form axial salt-bridges. By utilizing carbodiimide-mediated amidation between amine and carboxylate, the crosslinked collagen helical dimers and trimers were observed on MALDI-TOF MS. Lastly, we attempted to design customized CMPs for natural collagen targeting. Based on different sequences of host natural collagens, the guest CMPs contain substitutions to maximize pairwise interactions, including charge pairs and cation-pi pairs. The CMP design was demonstrated to form destabilized collagen helical structure, but failed to hybridize with the synthetic natural collagen. Through the exploration of different host peptides, we believe this strategy would provide an advanced model once a complete understanding of substitution effect in collagen is given.
Design, self-assembly and applications of heterotrimeric collagen mimics
(2009) Gauba, Varun; Hartgerink, Jeffrey D.
Collagen, a fibrous protein, is an essential structural component of all connective tissues, including cartilage, skin, tendon, ligaments and bone. Type I collagen is an AAB heterotrimer assembled from two identical alpha1 and one alpha2 chain. Missense mutations in either the alpha1 or alpha2 chains of type I collagen, which lead to the substitution of Gly in the ubiquitous X-Y-Gly repeat by bulky amino acids lead to Osteogenesis imperfecta (OI) of varying severity. However, the majority of studies on the effects of amino acid substitutions on triple helix stability have been performed on collagen-like peptides homotrimers. We report the design, synthesis, self-assembly and characterization of a series of peptides that self-assemble to form collagen-like heterotrimers directed through electrostatic interactions. First, we utilize a series of peptides with net charge ranging from -10 to +10 to show the assembly of various AAB and ABC heterotrimers. We then analyze the ability of various charge pairs based upon naturally occurring amino acids, for instance E--R, E--K, D--R and D--K charge pairs, to stabilize a collagen triple helix. We report the synthesis of a surprisingly stable ABC heterotrimer, composed of (DOG)10, (PKG)10 and (POG) 10 chains (O = hydroxyproline), with a stability comparable to (POG) 10 homotrimer. This high stability heterotrimer is then used to develop a peptide model for OI, a hereditary disorder observed in type I collagen. We report the design of a novel peptide model that can mimic glycine mutations in either of the alpha1 or alpha2 chains of type I collagen. This design utilizes an electrostatic recognition motif in three chains that can force the interaction of any three peptides, including AAA (all same) homotrimers, AAB (two same, one different) heterotrimers and ABC (all different) heterotrimers. The component peptides can be designed in such a way that the mutations are present in none, one, two or all three chains. We successfully report collagen mutants, for the first time, with the structure relevant to the native forms of OI. Furthermore, we are able to differentiate between four triple helices that differ from each other in the frequency of glycine mutations at a particular position. Thus, the ease of preparation of heterotrimers, coupled with our ability to separate single mutations, provides us with a tool to understand mutations in natural collagens that lead to various connective tissue disorders in general and OI in particular. We also introduce another peptide model based upon the ABC heterotrimer to understand the effect of proline hydroxylation and fluorination to the stability of a collagen triple helix, in a chain dependent manner.
Development of a Synthetic Toolkit for the Exploration of All-Hydrocarbon α-Helical Stapled Peptide Chemical Space via DNA-Encoded Chemical Libraries
(2020-04-23) Monty, Olivier Brian Cyril; Young, Damian W; Ball, Zachary T; Hartgerink, Jeffrey D.; Egan, Scott P.
The recognized attrition of drug discovery has been the central motivation behind the work described in the following chapters. Such decline in the rate of discovery has been partially blamed on the exhaustive exploration of part of chemical space by largely flat, sp2-rich chemotypes, which, until recently, made up most screening libraries. The exploration of new chemical space through the incorporation of three-dimensional chemotypes has been adopted as a potential solution, which has proven effective as evidenced by the recent surge in three-dimensional molecular probes and therapeutics. All-hydrocarbon α-helical stapled peptides represent a class of such three-dimensional chemotypes that have proven capable of targeting the challenging intracellular protein-protein interactions (~80% of the proteome). Those cell-permeable mini-proteins have promoted peptide drug discovery to the main stage and the work described in this document is intended to buttress and exploit this rise to prominence. Because the chemical space of stapled peptides is relatively underexplored and due to the pressing need for new therapeutics in the face of global issues such as drug resistance, fast and deep exploration via the large numbers and diversity attainable in DNA-encoded chemistry is desirable. To do so, however, one needs a DNA-compatible synthetic toolkit to generate all-hydrocarbon staples on DNA-chemical conjugates and to perform peptide synthesis on DNA. Chapters 1 and 2 describe the development of such a toolkit. ii Chapter 3 finally discusses how the latter could be used for the exploration of the α-helical stapled peptide chemical space via DNA-encoded chemical libraries.
Drug-Triggered and Cross-Linked Self-Assembling Nanofibrous Hydrogels
(American Chemical Society, 2015) Kumar, Vivek A.; Shi, Siyu; Wang, Benjamin K.; Li, I-Che; Jalan, Abhishek A.; Sarkar, Biplab; Wickremasinghe, Navindee C.; Hartgerink, Jeffrey D.
Self-assembly of multidomain peptides (MDP) can be tailored to carry payloads that modulate the extracellular environment. Controlled release of growth factors, cytokines, and small-molecule drugs allows for unique control of in vitro and in vivo responses. In this study, we demonstrate this process of ionic cross-linking of peptides using multivalent drugs to create hydrogels for sustained long-term delivery of drugs. Using phosphate, heparin, clodronate, trypan, and suramin, we demonstrate the utility of this strategy. Although all multivalent anions result in good hydrogel formation, demonstrating the generality of this approach, suramin led to the formation of the best hydrogels per unit concentration and was studied in greater detail. Suramin ionically cross-linked MDP into a fibrous meshwork as determined by scanning and transmission electron microscopy. We measured material storage and loss modulus using rheometry and showed a distinct increase in G′ and G″ as a function of suramin concentration. Release of suramin from scaffolds was determined using UV spectroscopy and showed prolonged release over a 30 day period. Suramin bioavailability and function were demonstrated by attenuated M1 polarization of THP-1 cells compared to positive control. Overall, this design strategy has allowed for the development of a novel class of polymeric delivery vehicles with generally long-term release and, in the case of suramin, cross-linked hydrogels that can modulate cellular phenotype.
Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel
(American Chemical Society, 2023) Pogostin, Brett H.; Saenz, Gabriel; Cole, Carson C.; Euliano, Erin M.; Hartgerink, Jeffrey D.; McHugh, Kevin J.
Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.
Engineering Adeno-Associated Virus for Protease Targeted Gene Therapy and Immune Avoidance
(2019-04-19) Robinson, Tawana M; Hartgerink, Jeffrey D.; Suh, Junghae
Adeno-associated virus (AAV) has earned significant attention as a safe and efficient gene therapy tool. AAV has been used in over 100 clinical trials to treat a variety of human diseases. However, non-specific targeting to diseased cells and activation of the host immune response hinder its therapeutic efficacy. To address these challenges, genetic modification of the AAV capsid can lead to an improved gene delivery platform. Therefore, capsid-engineering strategies may be necessary to develop optimized vectors for clinical progress. This present work reveals design rules governed by amino acid properties for engineered AAV to become activated by upregulated proteolytic biomarkers in diseased sites. AAV constructs with varying chemical properties were synthesized and characterized for functional behavior. In parallel, a Nature-inspired strategy was employed to create an immune-evasive AAV vector. A panel of AAV vectors with inserted stealth peptides in the AAV capsid was generated to study immune cell uptake. Finally, to gain a better understanding of AAV intracellular trafficking, we found several amino acid residues that are necessary for viral infectivity. The ultimate goal for my research contributions is to develop and to advance AAV vectors for future clinical applications.
Evaluating the physicochemical effects of conjugating peptides into thermogelling hydrogels for regenerative biomaterials applications
(Oxford University Press, 2021) Pearce, Hannah A.; Jiang, Emily Y.; Swain, Joseph W.R.; Navara, Adam M.; Guo, Jason L.; Kim, Yu Seon; Woehr, Andrew; Hartgerink, Jeffrey D.; Mikos, Antonios G.
Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study’s aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.
Highly Angiogenic Peptide Nanofibers
(American Chemical Society, 2015) Kumar, Vivek A.; Taylor, Nichole L.; Shi, Siyu; Wang, Benjamin K.; Jalan, Abhishek A.; Kang, Marci K.; Wickremasinghe, Navindee C.; Hartgerink, Jeffrey D.
Major limitations of current tissue regeneration approaches using artificial scaffolds are fibrous encapsulation, lack of host cellular infiltration, unwanted immune responses, surface degradation preceding biointegration, and artificial degradation byproducts. Specifically, for scaffolds larger than 200–500 μm, implants must be accompanied by host angiogenesis in order to provide adequate nutrient/waste exchange in the newly forming tissue. In the current work, we design a peptide-based self-assembling nanofibrous hydrogel containing cell-mediated degradation and proangiogenic moieties that specifically address these challenges. This hydrogel can be easily delivered by syringe, is rapidly infiltrated by cells of hematopoietic and mesenchymal origin, and rapidly forms an extremely robust mature vascular network. Scaffolds show no signs of fibrous encapsulation and after 3 weeks are resorbed into the native tissue. These supramolecular assemblies may prove a vital paradigm for tissue regeneration and specifically for ischemic tissue disease.
Hydrogel delivery of sting immunotherapy for treatment cancer
(2024-04-30) Young, Simon; Leach, David; Hartgerink, Jeffrey D.; Rice University; The Board of Regents of the University of Texas System; United States Patent and Trademark Office
In one aspect, the present disclosure provides for novel compositions of matter comprising multi domain peptide (MDP) hydrogels and cyclic dinucleotides (CDNs). Also disclosed are method of using such compositions in the treatment of cancer, including in particular the treatment of head and neck cancers, such as those resistant to CDN therapy.
Hydroxyproline-free Single Composition ABC Collagen Heterotrimer
(American Chemical Society, 2013) Jalan, Abhishek A.; Demeler, Borries; Hartgerink, Jeffrey D.
Hydroxyproline plays a major role in stabilizing collagenous domains in eukaryotic organisms. Lack of this modification is associated with significant lowering in thermal stability of the collagen triple helix and may also affect fibrillogenesis and folding of the peptide chains. In contrast, even though bacterial collagens lack hydroxyproline, their thermal stability is comparable to fibrillar collagen. This has been attributed to the high frequency of charged amino acids found in bacterial collagen. Here we report a thermally stable hydroxyproline-free ABC heterotrimeric collagen mimetic system composed of decapositive and decanegative peptides and a zwitterionic peptide. None of the peptides contain hydroxyproline and furthermore the zwitterionic peptide does not even contain proline. The heterotrimer is electrostatically stabilized via multiple interpeptide lysine-aspartate and lysine-glutamate salt-bridges and maintains good thermal stability with a melting temperature of 37 °C. The ternary peptide mixture also populates a single composition ABC heterotrimer as confirmed by circular dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy. This system illustrates the power of axial salt-bridges to direct and stabilize the self-assembly of a triple helix and may be useful in analogous designs in expression systems where the incorporation of hydroxyproline is challenging.
I. Charged pair hydrogen bonding interactions in collagen heterotrimers. II. Surface enhanced Raman spectroscopy of aromatic peptides
(2010) Wei, Fang; Hartgerink, Jeffrey D.
Eight ABC heterotrimers whose self-assembly are directed through electrostatic interactions were studied here. Oppositely charged pairs of amino acids, with varying side chain length, were assessed for their ability to stabilize a triple helix. Aspartate-lysine was found to result in the most thermally stable helix followed by lysine-glutamate, ornithine-aspartate and finally ornithine-glutamate. When the sequence position of these charged amino acids was reversed from what is normally observed in nature, triple helix stability and compositional purity was significantly reduced. The effect of salt on triple helix stability was explored and it was observed that increased salt concentration reduces the thermal stability of heterotrimers by an average of 5°C, but does not disrupt helix assembly. It was also found that positively charged homotrimers can be stabilized in the presence of phosphate anions. Raman and Surface-enhanced Raman spectroscopies (SERS) are potentially important tools in the characterization of biomolecules such as proteins and DNA. In this work, SERS spectra of three cysteine containing aromatic peptides: tryptophan-cysteine, tyrosine-cysteine, and phenylalanine-cysteine, bound to Au nanoshell substrates, were obtained and compared to their respective normal Raman spectra. While the full widths at half maximum of the SERS peaks are significantly broadened (up to 70%), no significant spectral shifts (<6 cm-1) of the major Stokes modes were observed between the two modalities. It is shown that the Raman and SERS spectra of penetratin, a cell-penetrating peptide, can be evaluated quite reliably from the spectra of its constituent aromatic amino acids except in the -CH2- bending and amide I and III regions where the spectral intensities are critically dependent on the chain length and/or protein conformations. From this study we conclude that with aromatic amino acid residues provide the overwhelmingly dominant features in the Raman and SERS spectra of peptides and proteins, and that the Raman modes of these three small constructed peptides can apply directly to the assignment of Raman and SERS features in the spectra of larger peptides and proteins.
Local Anti–PD-1 Delivery Prevents Progression of Premalignant Lesions in a 4NQO-Oral Carcinogenesis Mouse Model
(American Association for Cancer Research, 2021) Shi, Yewen; Xie, Tong-xin; Leach, David G.; Wang, Bingbing; Young, Simon; Osman, Abdullah A.; Sikora, Andrew G.; Ren, Xiaoyong; Hartgerink, Jeffrey D.; Myers, Jeffrey N.; Rangel, Roberto
Although the principle of systemic treatment to prevent the progression of oral premalignant lesions (OPL) has been demonstrated, there remains a lack of consensus about an optimal approach that balances clinical efficacy with toxicity concerns. Recent advances in cancer therapy using approaches targeting the tumor immune microenvironment (TIME) including immune-checkpoint inhibitors indicate that these agents have significant clinically activity against different types of cancers, including oral cancer, and therefore they may provide an effective oral cancer prevention strategy for patients with OPLs. Our past work showed that systemic delivery of a monoclonal antibody to the programmed death receptor 1 (PD-1) immune checkpoint can inhibit the progression of OPLs to oral cancer in a syngeneic murine oral carcinogenesis model. Here we report a novel approach of local delivery of a PD-1 immune-checkpoint inhibitor loaded using a hydrogel, which significantly reduces the progression of OPLs to carcinomas. In addition, we detected a significant infiltration of regulatory T cells associated with oral lesions with p53 mutation, and a severe loss of expression of STING, which correlated with a decreased infiltration of dendritic cells in the oral lesions. However, a single local dose of PD-1 inhibitor was found to restore stimulator of interferon response cGAMP interactor 1 (STING) and CD11c expression and increase the infiltration of CD8+ T cells into the TIME irrespective of the p53 mutational status. Overall, we provide evidence for the potential clinical value of local delivery of biomaterials loaded with anti–PD-1 antibodies to prevent malignant progression of OPLs. Prevention Relevance: Oral cancer is an aggressive disease, with an overall survival rate of 50%. Preinvasive histologic abnormalities such as tongue dysplasia represent an early stage of oral cancer; however, there are no treatments to prevent oral carcinoma progression. Here, we combined biomaterials loaded with an immunotherapeutic agent preventing oral cancer progression.

Browsing by Author "Hartgerink, Jeffrey D."

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