Recent Advances in Bioorthogonal Click Chemistry for Biomedical Applications.

Bioorthogonal click chemistry, first introduced in the early 2000s, has become one of the most widely used approaches for designing advanced biomaterials for applications in tissue engineering and regenerative medicine, due to the selectivity and biocompatibility of the associated reactants and reaction conditions. In this review, we present recent advances in utilizing bioorthogonal click chemistry for the development of three-dimensional, biocompatible scaffolds and cell-encapsulated biomaterials. Additionally, we highlight recent examples using these approaches for biomedical applications including drug delivery, imaging, and cell therapy and discuss their potential as next generation biomaterials.

Peptoid nanosheets exhibit a new secondary-structure motif.

A promising route to the synthesis of protein-mimetic materials that are capable of complex functions, such as molecular recognition and catalysis, is provided by sequence-defined peptoid polymers--structural relatives of biologically occurring polypeptides. Peptoids, which are relatively non-toxic and resistant to degradation, can fold into defined structures through a combination of sequence-dependent interactions. However, the range of possible structures that are accessible to peptoids and other biological mimetics is unknown, and our ability to design protein-like architectures from these polymer classes is limited. Here we use molecular-dynamics simulations, together with scattering and microscopy data, to determine the atomic-resolution structure of the recently discovered peptoid nanosheet, an ordered supramolecular assembly that extends macroscopically in only two dimensions. Our simulations show that nanosheets are structurally and dynamically heterogeneous, can be formed only from peptoids of certain lengths, and are potentially porous to water and ions. Moreover, their formation is enabled by the peptoids' adoption of a secondary structure that is not seen in the natural world. This structure, a zigzag pattern that we call a Σ('sigma')-strand, results from the ability of adjacent backbone monomers to adopt opposed rotational states, thereby allowing the backbone to remain linear and untwisted. Linear backbones tiled in a brick-like way form an extended two-dimensional nanostructure, the Σ-sheet. The binary rotational-state motif of the Σ-strand is not seen in regular protein structures, which are usually built from one type of rotational state. We also show that the concept of building regular structures from multiple rotational states can be generalized beyond the peptoid nanosheet system.

Design and preparation of organic nanomaterials using self-assembled peptoids.

The self-assembly and self-organization of peptoids, peptidomimetic polymers composed of N-substituted glycine monomers, can result in a plethora of well-defined organic nanostructures. Such classes of nanomaterials represent highly interesting functional platforms for many applications, for example, drug delivery, sensing, and catalysis. The main advantages of using self-assembling peptoids to engineer organic nanostructures include their chemical diversity, biocompatibility, enzymatic stability, and ease of synthesis. The goal of this review is to present a comprehensive summary of the most relevant studies regarding the self-assembling process of peptoids into zero-, one-, and two-dimensional nanostructures, with a focus on their mechanism of formation and their potential applications.

Design, Synthesis, Assembly, and Engineering of Peptoid Nanosheets.

Two-dimensional (2D) atomically defined organic nanomaterials are an important material class with broad applications. However, few general synthetic methods exist to produce such materials in high yields and to precisely functionalize them. One strategy to form ordered 2D organic nanomaterials is through the supramolecular assembly of sequence-defined synthetic polymers. Peptoids, one such class of polymer, are designable bioinspired heteropolymers whose main-chain length and monomer sequence can be precisely controlled. We have recently discovered that individual peptoid polymers with a simple sequence of alternating hydrophobic and ionic monomers can self-assemble into highly ordered, free-floating nanosheets. A detailed understanding of their molecular structure and supramolecular assembly dynamics provides a robust platform for the discovery of new classes of nanosheets with tunable properties and novel applications. In this Account, we discuss the discovery, characterization, assembly, molecular modeling, and functionalization of peptoid nanosheets. The fundamental properties of peptoid nanosheets, their mechanism of formation, and their application as robust scaffolds for molecular recognition and as templates for the growth of inorganic minerals have been probed by an arsenal of experimental characterization techniques (e.g., scanning probe, electron, and optical microscopy, X-ray diffraction, surface-selective vibrational spectroscopy, and surface tensiometry) and computational techniques (coarse-grained and atomistic modeling). Peptoid nanosheets are supramolecular assemblies of 16-42-mer chains that form molecular bilayers. They span tens of microns in lateral dimensions and freely float in water. Their component chains are highly ordered, with chains nearly fully extended and packed parallel to one another as a result of hydrophobic and electrostatic interactions. Nanosheets form via a novel interface-catalyzed monolayer collapse mechanism. Peptoid chains first assemble into a monolayer at either an air-water or oil-water interface, on which peptoid chains extend, order, and pack into a brick-like pattern. Upon mechanical compression of the interface, the monolayer buckles into stable bilayer structures. Recent work has focused on the design of nanosheets with tunable properties and functionality. They are readily engineerable, as functional monomers can be readily incorporated onto the nanosheet surface or into the interior. For example, functional hydrophilic "loops" have been displayed on the surfaces of nanosheets. These loops can interact with specific protein targets, serving as a potentially general platform for molecular recognition. Nanosheets can also bind metal ions and serve as 2D templates for mineral growth. Through our understanding of the formation mechanism, along with predicted features ascertained from molecular modeling, we aim to further design and synthesize nanosheets as robust protein mimetics with the potential for unprecedented functionality and stability.

Ammonium and guanidinium dendron-carbon nanotubes by amidation and click chemistry and their use for siRNA delivery.

A series of multi-walled carbon nanotube (MWCNT) conjugates is described, functionalized with different dendrons bearing positive charges at their termini (i.e. ammonium or guanidinium groups). The dendrimeric units are anchored to the nanotube scaffolds using two orthogonal synthetic approaches, amidation and click reactions. The final nanohybrids are characterized by complementary analytical techniques, while their ability to interact with siRNA is investigated by means of agarose gel electrophoresis. The demonstration of the cell uptake capacity, the low cytotoxicity, and the ability of these cationic conjugates to silence cytotoxic genes suggests them to be promising carriers for genetic material.

Inducing mesenchymal stem cell attachment on non-cell adhesive hydrogels through click chemistry.

We introduce an innovative approach to adhere mesenchymal stem cells (MSCs) to a hydrogel scaffold by nucleating adhesion through strain-promoted click chemistry. This method yields a significant increase in cell viability compared to non-functionalized and RGD peptide functionalized hydrogels, providing a promising alternative to traditional biomaterials cell attachment approaches.

Controlled delivery of a protein tyrosine phosphatase inhibitor, SHP099, using cyclodextrin-mediated host-guest interactions in polyelectrolyte multilayer films for cancer therapy.

The Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) is a key enzyme in pathways regulating tumor growth signaling, and recently gained interest as a promising anticancer drug target. Many SHP2 inhibitors are currently under development, including SHP099, which has shown potent anticancer activity at low concentrations in vivo. In this work, we developed multilayer coatings for localized delivery of SHP099 to improve upon current cancer therapies. Layer-by-layer self-assembly was used to develop films composed of chitosan and poly-carboxymethyl-ß-cyclodextrin (PßCD) for the delivery of SHP099. SHP099 was successfully loaded into multilayer films via host-guest interactions with PßCD. Nuclear magnetic resonance spectroscopy confirmed the occurrence of this supramolecular assembly by identifying the interaction of specific terminal SHP099 protons with the protons of the CD. SHP099 release from assembled films was detected over 96 hours, and was found to inhibit colony formation of human breast adenocarcinoma cells in vitro. These multilayer films have the potential to be used in a range of anticancer applications and overcome common complications of systemic chemotherapeutic administration, while maximizing SHP099 efficacy.

Glycosylated Peptoid Nanosheets as a Multivalent Scaffold for Protein Recognition.

Glycoproteins adhered on the cellular membrane play a pivotal role in a wide range of cellular functions. Their importance is particularly relevant in the recognition process between infectious pathogens (such as viruses, bacteria, toxins) and their host cells. Multivalent interactions at the pathogen-cell interfaces govern binding events and can result in a strong and specific interaction. Here we report an approach to mimic the cell surface presentation of carbohydrate ligands by the multivalent display of sugars on the surface of peptoid nanosheets. The constructs provide a highly organized 2D platform for recognition of carbohydrate-binding proteins. The sugars were displayed using different linker lengths or within loops containing 2-6 hydrophilic peptoid monomers. Both the linkers and the loops contained one alkyne-bearing monomer, to which different saccharides were attached by copper-catalyzed azide-alkyne cycloaddition reactions. Peptoid nanosheets functionalized with different saccharide groups were able to selectively bind multivalent lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by fluorescence microscopy and a homogeneous Förster resonance energy transfer (FRET)-based binding assay. To evaluate the potential of this system as sensor for threat agents, the ability of functionalized peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets were functionalized with globotriose, the natural ligand of Shiga toxin, and the effective binding of the nanomaterial was verified by the FRET-based binding assay. In all cases, evidence for multivalent binding was observed by systematic variation of the ligand display density on the nanosheet surface. These cell surface mimetic nanomaterials may find utility in the inactivation of pathogens or as selective molecular recognition elements.

Evidence for cis Amide Bonds in Peptoid Nanosheets.

Peptoid nanosheets are supramolecular protein-mimetic materials that form from amphiphilic polypeptoids with aromatic and ionic side chains. Nanosheets have been studied at the nanometer scale, but the molecular structure has been difficult to probe. We report the use of 13C-13C dipolar recoupling solid-state NMR measurements to reveal the configuration of backbone amide bonds selected by 13C isotopic labeling of adjacent α-carbons. Measurements on the same molecules in the amorphous state and in nanosheets revealed that amide bonds in the center of the amino block of peptoid (NaeNpe)7-(NceNpe)7 (B28) favor the trans configuration in the amorphous state and the cis configuration in the nanosheet. This unexpected result contrasts with previous NMR and theoretical studies of short solvated peptoids. Furthermore, examination of the amide bond at the junction of the two charged blocks within B28 revealed a mixture of both cis and trans configurational states, consistent with the previously predicted brickwork-like intermolecular organization.

Improved chemical and mechanical stability of peptoid nanosheets by photo-crosslinking the hydrophobic core.

Peptoid nanosheets can be broadly functionalized for a variety of applications. However, they are susceptible to degradation when exposed to chemical or mechanical stress. To improve their strength, photolabile monomers were introduced in order to crosslink the nanosheet interior. Photo-crosslinking produced a more robust material that can survive sonication, lyophilization, and other biochemical manipulations.

Carbon nanomaterials as new tools for immunotherapeutic applications.

Carbon-based nanomaterials, including carbon nanotubes and graphene, have gained great attention in the scientific community due to their unique physico-chemical properties, which could be also promising in many biomedical-related fields. In particular, their low cytotoxicity, achieved when properly functionalized, along with the possibility to link multiple bioactive molecules, realistically allows envisaging their potential use as a therapeutic platform. In this context, the immune system and immune responses play an important role in our organism, as they are involved either directly or indirectly in many diseases. Therefore, the possibility to prevent or block a disease by controlling and/or modulating the immune responses has become an important task in nanomedicine. In this feature article the advantages of using carbon-based materials in immunotherapy are presented. Important goals achieved using carbon nanotubes and graphene are described, highlighting the promising use of these nanomaterials in cancer treatment, imaging and vaccine development. The capacity of functionalized carbon nanotubes to modulate the immune responses is also discussed, highlighting the current state of the art and the future developments on this subject.

Endowing carbon nanotubes with biological and biomedical properties by chemical modifications.

The scope of nanotechnology is gaining importance in biology and medicine. Carbon nanotubes (CNTs) have emerged as a promising tool due to their unique properties, high specific surface area, and capacity to cross biological barriers. These properties offer a variety of opportunities for applications in nanomedicine, such as diagnosis, disease treatment, imaging, and tissue engineering. Nevertheless, pristine CNTs are insoluble in water and in most organic solvents; thereby functionalization of their surface is necessary to increase biocompatibility. Derivatization of CNTs also gives the possibility to conjugate different biological and bioactive molecules including drugs, proteins, and targeting ligands. This review focuses on the chemical modifications of CNTs that have been developed to impart specific properties for biological and medical purposes. Biomolecules can be covalently grafted or non-covalently adsorbed on the nanotube surface. In addition, the inner core of CNTs can be exploited to encapsulate drugs, nanoparticles, or radioactive elements.

Double functionalization of carbon nanotubes with purine and pyrimidine derivatives.

Herein, we have developed a synthetic strategy for the covalent double functionalization of single-walled carbon nanotubes (SWCNTs) with a combination of purine-pyrimidine and purine-purine nucleobase systems. The nucleobases were introduced on the sidewall of oxidized SWCNTs through 1,3-dipolar cycloaddition and by amidation of the carboxylic acids located at the tips and defect sites of the nanotubes. The new nanohybrids were characterized by transmission electron microscopy, thermogravimetric analysis, FTIR and Raman spectroscopy, magic-angle spinning NMR spectroscopy, and Kaiser test. The nucleobase/SWCNT conjugates can be envisaged for the modulation of the interactions with nucleic acids by means of base pairing, thereby opening new possibilities in the development of DNA/CNT nanobioconjugates.

Peptide-based carbon nanotubes for mitochondrial targeting.

In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TEM.