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Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Here, we study the upper critical solution temperature triggered phase transition of thermally responsive poly(ethylene glycol)-block-poly(ethylene glycol) methyl ether acrylate-co-poly(ethylene glycol) phenyl ether acrylate-block-polystyrene nanoassemblies in isopropanol. To gain mechanistic insight into the organic solution-phase dynamics of the upper critical solution temperature polymer, we leverage variable temperature liquid-cell transmission electron microscopy correlated with variable temperature liquid resonant soft X-ray scattering. Heating above the upper critical solution temperature triggers a reduction in particle size and a morphological transition from a spherical core shell particle with a complex, multiphase core to a micelle with a uniform core and Gaussian polymer chains attached to the surface. These correlated solution phase methods, coupled with mass spectral validation and modeling, provide unique insight into these thermoresponsive materials. Moreover, we detail a generalizable workflow for studying complex, solution-phase nanomaterials via correlative methods.

A simple strategy for generating stimuli-responsive peptide-based hydrogels via charge-conversion of a self-assembling peptide (SAP) is described. These materials are formulated as soluble, polyanionic peptides, containing maleic acid, citraconic acid, or dimethylmaleic acid masking groups on each lysine residue, which do not form assemblies, but instead flow easily through high gauge needles and catheters. Acid-induced mask hydrolysis renews the zwitterionic nature of the peptides with concomitant and rapid self-assembly via β-sheet formation into rehealable hydrogels. The use of different masks enables one to tune pH responsiveness and assembly kinetics. In anticipation of their potential for in vivo hydrogel delivery and use, progelators exhibit hemocompatibility in whole human blood, and their peptide components are shown to be noncytotoxic. Finally, demonstration of stimuli-induced self-assembly for dye sequestration suggests a simple, non-covalent strategy for small molecule encapsulation in a degradable scaffold. In summary, this simple, scalable masking strategy allows for preparation of responsive, dynamic self-assembling biomaterials. This work sets the stage for implementing biodegradable therapeutic hydrogels that assemble in response to physiological, disease-relevant states of acidosis.

In this report, we explore the internal structural features of polyMOFs consisting of equal mass ratios of metal-coordinating poly(benzenedicarboxylic acid) blocks and non-coordinating poly(ethylene glycol) (PEG) blocks. The studies reveal alternating lamellae of metal-rich, crystalline regions and metal-deficient non-crystalline polymer, which span the length of hundreds of nanometers. Polymers consisting of random PEG blocks, PEG end-blocks, or non-coordinating poly(cyclooctadiene) (COD) show similar alternation of metal-rich and metal-deficient regions, indicating a universal self-assembly mechanism. A variety of techniques were employed to interrogate the internal structure of the polyMOFs, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and small-angle synchrotron X-ray scattering (SAXS). Independent of the copolymer architecture or composition, the internal structure of the polyMOF crystals showed similar lamellar self-assembly at single-nanometer length scales.

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. Here we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are demonstrated by preparing patterns from combinatorial polymer and block copolymer brushes. Images from polymeric pixels are created using the light reflected from a DMD to photochemically initiate atom-transfer radical polymerization from initiators immobilized on Si/SiO2 wafers. Patterning is combined with high-throughput analysis of grafted-from polymerization kinetics, accelerating reaction discovery, and optimization of polymer coatings.

We describe cyclic peptide progelators which cleave in response to UV light to generate linearized peptides which then self-assemble into gel networks. Cyclic peptide progelators were synthesized, where the peptides were sterically constrained, but upon UV irradiation, predictable cleavage products were generated. Amino acid sequences and formulation conditions were altered to tune the mechanical properties of the resulting gels. Characterization of the resulting morphologies and chemistry was achieved through liquid phase and standard TEM methods, combined with matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

Contrast-enhanced ultrasound (CEUS) offers the exciting prospect of retaining the ease of ultrasound imaging while enhancing imaging clarity, diagnostic specificity, and theranostic capability. To advance the capabilities of CEUS, the synthesis and understanding of new ultrasound contrast agents (UCAs) is a necessity. Many UCAs are nano- or micro-scale materials composed of a perfluorocarbon (PFC) and stabilizer that synergistically induce an ultrasound response that is both information-rich and easily differentiated from natural tissue. In this work, we probe the extent to which CEUS is modulated through variation in a PFC stabilized with fluorine-modified polydopamine nanoparticles (PDA NPs). The high level of synthetic tunability in this system allows us to study signal as a function of particle aggregation and PFC volatility in a systematic manner. Separation of aggregated and non-aggregated nanoparticles lead to a fundamentally different signal response, and for this system, PFC volatility has little effect on CEUS intensity despite a range of over 50 °C in boiling point. To further explore the imaging tunability and multimodality, Fe3+-chelation was employed to generate an enhanced photoacoustic (PA) signal in addition to the US signal. In vitro and in vivo results demonstrate that PFC-loaded PDA NPs show stronger PA signal than the non-PFC ones, indicating that the PA signal can be used for in situ differentiation between PFC-loading levels. In sum, these data evince the rich role synthetic chemistry can play in guiding new directions of development for UCAs.

We describe the design, synthesis, and antitumor activity of an 18 carbon α,ω-dicarboxylic acid monoconjugated via an ester linkage to paclitaxel (PTX). This 1,18-octadecanedioic acid-PTX (ODDA-PTX) prodrug readily forms a noncovalent complex with human serum albumin (HSA). Preservation of the terminal carboxylic acid moiety on ODDA-PTX enables binding to HSA in the same manner as native long-chain fatty acids (LCFAs), within hydrophobic pockets, maintaining favorable electrostatic contacts between the ω-carboxylate of ODDA-PTX and positively charged amino acid residues of the protein. This carrier strategy for small molecule drugs is based on naturally evolved interactions between LCFAs and HSA, demonstrated here for PTX. ODDA-PTX shows differentiated pharmacokinetics, higher maximum tolerated doses and increased efficacy in vivo in multiple subcutaneous murine xenograft models of human cancer, as compared to two FDA-approved clinical formulations, Cremophor EL-formulated paclitaxel (crPTX) and Abraxane (nanoparticle albumin-bound (nab)-paclitaxel).

Injectable biopolymer hydrogels have gained attention for use as scaffolds to promote cardiac function and prevent negative left ventricular (LV) remodeling post-myocardial infarction (MI). However, most hydrogels tested in preclinical studies are not candidates for minimally invasive catheter delivery due to excess material viscosity, rapid gelation times, and/or concerns regarding hemocompatibility and potential for embolism. We describe a platform technology for progelator materials formulated as sterically constrained cyclic peptides which flow freely for low resistance injection, and rapidly assemble into hydrogels when linearized by disease-associated enzymes. Their utility in vivo is demonstrated by their ability to flow through a syringe and gel at the site of MI in rat models. Additionally, synthetic functionalization enables these materials to flow through a cardiac injection catheter without clogging, without compromising hemocompatibility or cytotoxicity. These studies set the stage for the development of structurally dynamic biomaterials for therapeutic hydrogel delivery to the MI.

We describe the observation of stimuli-induced peptide-based nanoscale assemblies by liquid cell transmission electron microscopy (LCTEM). LCTEM offers the opportunity to directly image nanoscale materials in liquid. Despite broad interest in characterizing biological phenomena, electron beam-induced damage remains a significant problem. Concurrently, methods for verifying chemical structure during or following an LCTEM experiment have been few, with key examples limited to electron diffraction or elemental analysis of crystalline materials; this strategy is not translatable to biopolymers observed in nature. In this proof-of-concept study, oligomeric peptides are biologically or chemically stimulated within the liquid cell in a TEM to assemble into nanostructures. The resulting materials are analyzed by MALDI-imaging mass spectrometry (MALDI-IMS) to verify their identity. This approach confirms whether higher-order assemblies observed by LCTEM consist of intact peptides, verifying that observations made during the in situ experiment are because of those same peptides and not aberrant electron beam damage effects.

The discovery of peptide substrates for enzymes with exclusive, selective activities is a central goal in chemical biology. In this paper, we develop a hybrid computational and biochemical method to rapidly optimize peptides for specific, orthogonal biochemical functions. The method is an iterative machine learning process by which experimental data is deposited into a mathematical algorithm that selects potential peptide substrates to be tested experimentally. Once tested, the algorithm uses the experimental data to refine future selections. This process is repeated until a suitable set of de novo peptide substrates are discovered. We employed this technology to discover orthogonal peptide substrates for 4'-phosphopantetheinyl transferase, an enzyme class that covalently modifies proteins. In this manner, we have demonstrated that machine learning can be leveraged to guide peptide optimization for specific biochemical functions not immediately accessible by biological screening techniques, such as phage display and random mutagenesis.

In nanomedicine, determining the spatial distribution of particles and drugs, together and apart, at high resolution within tissues, remains a major challenge because each must have a different label or detectable feature that can be observed with high sensitivity and resolution. We prepared nanoparticles capable of enzyme-directed assembly of particle therapeutics (EDAPT), containing an analogue of the Pt(II)-containing drug oxaliplatin, an 15N-labeled monomer in the hydrophobic block of the backbone of the polymer, the near-infrared dye Cy5.5, and a peptide that is a substrate for tumor metalloproteinases in the hydrophilic block. When these particles reach an environment rich in tumor associated proteases, the hydrophilic peptide substrate is cleaved, causing the particles to accumulate through a morphology transition, locking them in the tumor extracellular matrix. To evaluate the distribution of drug and EDAPT carrier in vivo, the localization of the isotopically labeled polymer backbone was compared to that of Pt by nanoscale secondary ion mass spectrometry (NanoSIMS). The correlation of NanoSIMS with super-resolution fluorescence microscopy revealed the release of the drug from the nanocarrier and colocalization with cellular DNA within tumor tissue. The results confirmed the dependence of particle accumulation and Pt(II) drug delivery on the presence of a Matrix Metalloproteinase (MMP) substrate and demonstrated antitumor activity. We conclude that these techniques are powerful for the elucidation of the localization of cargo and carrier, and enable a high-resolution assessment of their performance following in vivo delivery.

Researchers report the synthesis of a new archaea-inspired tetraether lipid and study the mechanical elastic properties of membranes made from this bolalipid. To access structural diversity in the hydrophobic moiety of the lipids and to tune membrane properties, they have used used Huisgen 1,3-dipolar cycloaddition strategy to design and synthesize a tetraether lipid incorporating phytanyl side chains and polar 1,4 triazole rings known to change lipid packing. The researchers demonstrate the first example of the synthesis of phytanyl side chain incorporated tetraether lipids with 1,4-triazole rings. They also confirm the ability of lipids incorporating 1,4-triazole rings to form stable small and giant vesicles and examined their physical and mechanical properties using a micropipette aspiration technique.

The passive leakage of small molecules across membranes is a major limitation of liposomal drug formulations. Here, we evaluate the leakage of 3 clinically used chemotherapeutic agents (cytarabine, methotrexate and vincristine) encapsulated in liposomes comprised of a synthetic, archaea-inspired, membrane-spanning tetraether lipid. Liposomes comprised of the pure tetraether lipid exhibited superior retention of both a neutrally and positively charged drug (up to an ∼9-fold decrease in the rate of drug leakage) compared to liposomes formed from a commercial diacyl lipid, while exhibiting a similar retention of a negatively charged drug that did not appreciably leak from either type of liposome. We also demonstrate that liposomes made of the archaea-inspired lipid can be used for the delivery of encapsulated small molecules into living cells.

A Gd3+-coordinated polymerizable analogue of the MRI contrast agent Gd-DOTA was used to prepare amphiphilic block copolymers, with hydrophilic blocks composed entirely of the polymerized contrast agent. The resulting amphiphilic block copolymers assemble into nanoparticles (NPs) of spherical- or fibril-shape, each demonstrating enhanced relaxivity over Gd-DOTA. As an initial examination of their behavior in vivo, intraperitoneal (IP) injection of NPs into live mice was performed, showing long IP residence times, observed by MRI. Extended residence times for particles of well-defined morphology may represent a valuable design paradigm for treatment or diagnosis of peritoneal malignances.

The synthesis of functional polymers encoded with biomolecules has been an extensive area of research for decades. As such, a diverse toolbox of polymerization techniques and bioconjugation methods has been developed. The greatest impact of this work has been in biomedicine and biotechnology, where fully synthetic and naturally derived biomolecules are used cooperatively. Despite significant improvements in biocompatible and functionally diverse polymers, our success in the field is constrained by recognized limitations in polymer architecture control, structural dynamics, and biostabilization. This Perspective discusses the current status of functional biosynthetic polymers and highlights innovative strategies reported within the past five years that have made great strides in overcoming the aforementioned barriers.

Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights the great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.

Amphiphilic block copolymers can assemble into a variety of structures on the nanoscale in selective solvent. The micelle blending protocol offers a simple unique route to reproducibly produce polymer nanostructures. Here we expand this blending protocol to a range of polymer micelle systems and self-assembly routes. We found by exploring a range of variables that the systems must be able to reach global equilibrium at some point for the blending protocol to be successful. Our results demonstrate the kinetics requirements, specifically core block glass transition temperature, Tg, and length of the block limiting the exchange rates, for the blending protocol which can then be applied to a wide range of polymer systems to access this simple protocol for polymer self-assembly.

Amphiphilic triblock copolymers containing Fe(III) -catecholate complexes formulated as spherical- or cylindrical-shaped micellar nanoparticles (SMN and CMN, respectively) are described as new T1-weighted agents with high relaxivity, low cytotoxicity, and long-term stability in biological fluids. Relaxivities of both SMN and CMN exceed those of established gadolinium chelates across a wide range of magnetic field strengths. Interestingly, shape-dependent behavior is observed in terms of the particles' interactions with HeLa cells, with CMN exhibiting enhanced uptake and contrast via magnetic resonance imaging (MRI) compared with SMN. These results suggest that control over soft nanoparticle shape will provide an avenue for optimization of particle-based contrast agents as biodiagnostics. The polycatechol nanoparticles are proposed as suitable for preclinical investigations into their viability as gadolinium-free, safe, and effective imaging agents for MRI contrast enhancement.

The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets-all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere.

Polylactic acid (PLA) has found widespread use in plastics and in biomedical applications due to its biodegradability into natural benign products. However, PLA-based materials remain limited in usefulness due to difficulty of incorporating functional groups into the polymer backbone. In this paper, we report a strategy for PLA functionalization that establishes the preparation of highly derivatized materials in which ring opening metathesis polymerization (ROMP) is employed as a graft-from polymerization technique utilizing a norbornene-modified handle incorporated into the PLA backbone. As a demonstration of this new synthetic methodology, a PLA-derived nanoparticle bearing imidazole units protected with a photolabile group was prepared. The morphology of this material could be controllably altered in response to exposure of UV light or acidic pH as a stimulus. We anticipate that this graft-from approach to derivatization of PLA could find broad use in the development of modified, biodegradable PLA-based materials.

A method for targeting to and retaining intravenously injected nanoparticles at the site of acute myocardial infarction in a rat model is described. Enzyme-responsive peptide-polymer amphiphiles are assembled as spherical micellar nanoparticles, and undergo a morphological transition from spherical-shaped, discrete materials to network-like assemblies when acted upon by matrix metalloproteinases (MMP-2 and MMP-9), which are up-regulated in heart tissue post-myocardial infarction.

An enzyme-responsive, paclitaxel-loaded nanoparticle is described and assessed in vivo in a human fibrosarcoma murine xenograft. This work represents a proof-of-concept study demonstrating the utility of enzyme-responsive nanoscale drug carriers capable of targeted accumulation and retention in tumor tissue in response to overexpressed endogenous enzymes.

In this paper we present in situ transmission electron microscopy of synthetic polymeric nanoparticles with emphasis on capturing motion in a solvated, aqueous state. The nanoparticles studied were obtained from the direct polymerization of a Pt(II)-containing monomer. The resulting structures provided sufficient contrast for facile imaging in situ. We contend that this technique will quickly become essential in the characterization of analogous systems, especially where dynamics are of interest in the solvated state. We describe the preparation of the synthetic micellar nanoparticles together with their characterization and motion in liquid water with comparison to conventional electron microscopy analyses.

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.