Synthesizing biomaterials in living organisms.

Living organisms fabricate biomacromolecules such as DNA, RNA, and proteins by the self-assembly process. The research on the mechanism of biomacromolecule formation also inspires the exploration of in vivo synthesized biomaterials. By elaborate design, artificial building blocks or precursors can self-assemble or polymerize into functional biomaterials within living organisms. In recent decades, these so-called in vivo synthesized biomaterials have achieved extensive applications in cell-fate manipulation, disease theranostics, bioanalysis, cellular surface engineering, and tissue regeneration. In this review, we classify strategies for in vivo synthesis into non-covalent, covalent, and genetic types. The development of these approaches is based on the chemical principles of supramolecular chemistry and synthetic chemistry, biological cues such as enzymes and microenvironments, and the means of synthetic biology. By summarizing the design principles in detail, some insights into the challenges and opportunities in this field are provided to  enlighten further research.

Old Dog New Tricks: PLGA Microparticles as an Adjuvant for Insulin Peptide Fragment-Induced Immune Tolerance against Type 1 Diabetes.

Poly[lactic-co-(glycolic acid)] (PLGA) is arguably one of the most versatile synthetic copolymers used for biomedical applications. In vivo delivery of multiple substances including cells, pharmaceutical compounds, and antigens has been achieved by using PLGA-based micro-/nanoparticles although, presently, the exact biological impact of PLGA particles on the immune system remains controversial. Type 1 diabetes (T1D) is one subtype of diabetes characterized by the attack of immune cells against self-insulin-producing pancreatic islet cells. Considering the autoimmune etiology of T1D and the recent use of PLGA particles for eliciting desired immune responses in various aspects of immunotherapy, for the present study, a combination of Ins29-23 peptide (a known autoantigen of T1D) and PLGA microparticles was selected for T1D prevention assessment in nonobese diabetic (NOD) mice, a well-known animal model with spontaneous development of T1D. Thus, inoculation of PLGA microparticles + Ins29-23 completely prevented T1D development, significantly better than untreated controls and mice treated by either PLGA microparticles or Ins29-23 per se. Subsequent mechanistic investigation further revealed a facilitative role of PLGA microparticles in immune tolerance induction. In summary, our data demonstrate an adjuvant potential of PLGA microparticles in tolerance induction and immune remodulation for effective prevention of autoimmune diseases such as T1D.

Recombinant proteins as cross-linkers for hydrogelations.

Protein-based hydrogels are promising materials for tissue engineering and drug delivery due to the unique properties of proteins such as perfect polydispersity, exact control over monomer sequence, ability to fine-tune molecular-level biochemical interactions, etc. This tutorial review summarizes recent progress on the preparation of protein-based hydrogels and their applications. Typically, we introduce two strategies of covalent and non-covalent ones for the preparation of hydrogels. Hydrogels prepared by the covalent strategy are stable and can respond to the conformational change of proteins. They can be applied for cells encapsulation, screening of drug molecules and heavy metals, etc. Hydrogels formed by non-covalent interactions are injectable physical hydrogels. The simple mixing preparation strategy and fast gelation kinetics guarantee the homogeneous encapsulation of cells and therapeutic agents within them. Therefore, they have been widely applied for the delivery of bioactive components, regenerative medicine, etc. The challenges that remained in this field are also summarized in this paper. We envision that rationally designed protein-based hydrogels will have broad applications in many areas including controlled delivery, tissue engineering, drug screening, etc.

Enzyme-instructed self-assembly (EISA) assists the self-assembly and hydrogelation of hydrophobic peptides.

Enzyme-instructed self-assembly (EISA) has several advantages in the preparation of supramolecular self-assembly materials for biomedical applications. In this study, we demonstrated that the enzyme-instructed self-assembly (EISA) strategy could assist the self-assembly and hydrogelation of two hydrophobic and bioactive peptides, tyroservatide (YSV) and laminin pentapeptide (YIGSR). We first synthesized the peptide derivatives of Nap-GFFYSV (peptide 1) and Nap-GFFYIGSR (peptide 2) and found that both peptides could not self-assemble into hydrogels due to their poor solubility. We therefore designed the phosphorylated precursors of the two hydrophobic peptides, Nap-GFFpYSV (precursor 1) and Nap-GFFpYIGSR (precursor 2), respectively, which had good solubility and can be dephosphorylated by alkaline phosphatase (ALP) to form supramolecular hydrogels. In addition, we found that the EISA could also occur on the surface of cells that overexpress ALP. The EISA strategy was a powerful method to generate hydrogels of hydrophobic compounds. We envision the big promise of the strategy in the preparation of biomaterials and nanomaterials of hydrophobic bioactive molecules.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells.

Amelogenin can induce odontogenic differentiation of human dental pulp cells (HDPCs), which has great potential and advantages in dentine-pulp complex regeneration. However, the unstability of amelogenin limits its further application. This study constructed amelogenin self-assembling peptide hydrogels (L-gel or D-gel) by heating-cooling technique, investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism. The critical aggregation concentration, conformation, morphology, mechanical property and biological stability of the hydrogels were characterized, respectively. The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement, quantitative reverse transcription polymerase chain reaction, western blot, Alizarin red staining and scanning electron microscope. The mechanism was explored via signaling pathway experiments. Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14, while the D-gel showed the highest enhancement effects. Meanwhile, the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21. The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways. Overall, our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization, which held big potential in the dentine-pulp complex regeneration.

Enzyme-triggered self-assembly of a small molecule: a supramolecular hydrogel with leaf-like structures and an ultra-low minimum gelation concentration.

We report on the use of a phosphatase to assist the formation of leaf-like structures and a supramolecular hydrogel with an ultra-low minimum gelation concentration. The compound can gel water at a minimum gelation concentration of 0.01 wt%, which is the lowest gelation concentration reported up to now. The images obtained by transmission electron microscopy (TEM) reveal the existence of leaf-like structures serving as the matrix of the hydrogels. The stability of the hydrogels was studied and emission spectra were used to get information about the molecular packing in the leaf-like structures. Since lowering the concentration of the gelator decreases the toxicity of the resulting hydrogels, ultra-low concentration gels have potential uses as biocompatible biomaterials for, e.g., cell cultures, tissue engineering, and drug delivery.

Delivery of MSCs with a Hybrid ß-Sheet Peptide Hydrogel Consisting IGF-1C Domain and D-Form Peptide for Acute Kidney Injury Therapy.

PURPOSE: By providing a stem cell microenvironment with particular bioactive constituents in vivo, synthetic biomaterials have been progressively successful in stem cell-based tissue regeneration by enhancing the engraftment and survival of transplanted cells. Designs with bioactive motifs to influence cell behavior and with D-form amino acids to modulate scaffold stability may be critical for the development and optimization of self-assembling biomimetic hydrogel scaffolds for stem cell therapy. MATERIALS AND METHODS: In this study, we linked naphthalene (Nap) covalently to a short D-form peptide (Nap-DFDFG) and the C domain of insulin-like growth factor-1 (IGF-1C) as a functional hydrogel-based scaffolds, and we hypothesized that this hydrogel could enhance the therapeutic efficiency of human placenta-derived mesenchymal stem cells (hP-MSCs) in a murine acute kidney injury (AKI) model. RESULTS: The self-assembling peptide was constrained into a classical ß-sheet structure and showed hydrogel properties. Our results revealed that this hydrogel exhibited increased affinity for IGF-1 receptor. Furthermore, cotransplantation of the ß-IGF-1C hydrogel and hP-MSCs contributed to endogenous regeneration post-injury and boosted angiogenesis in a murine AKI model, leading to recovery of renal function. CONCLUSION: This hydrogel could provide a favorable niche for hP-MSCs and thereby rescue renal function in an AKI model by promoting cell survival and angiogenesis. In conclusion, by covalently linking the desired functional groups to D-form peptides to create functional hydrogels, self-assembling ß-sheet peptide hydrogels may serve as a promising platform for tissue-engineering and stem cell therapy.

Supramolecular hydrogel of a D-amino acid dipeptide for controlled drug release in vivo.

A supramolecular hydrogel based on D-amino acids, which resists hydrolysis catalyzed by proteinase K and offers long-term biostability, exhibits controlled release in vivo, as proved by the pharmacokinetics of encapsulated 125I tracers and the SPECT imaging of the hydrogel-encapsulated 131I tracers. As the first in vivo imaging investigation of the drug release properties of the supramolecular hydrogel, isotope encapsulation serves as a valid, useful assay for characterizing the controlled release properties of supramolecular hydrogels in vivo. Our results indicate that supramolecular hydrogels promise new biomaterials for controlled drug release.

A supramolecular hydrogel to boost the production of antibodies for phosphorylated proteins.

Antibodies are widely used both in clinical practice and in research. However, the development of methods to increase the ratio of antibodies to recognize phosphorylated proteins remains challenging. In this study, we report a novel and useful method for the efficient production of antibodies for phosphorylated proteins. Based on our previously developed vaccine adjuvant Nap-GDFDFDY, we prepared hydrogels by the Ca2+-induced self-assembly of a phosphorylated peptide gelator Nap-GDFDFpDY. The hydrogel could protect phosphorylated antigens from being dephosphorylated by endogenous phosphatase, thus selectively increasing the ratio of the antibodies for phosphorylated proteins. Our study provides a useful strategy for the production of antibodies to recognize proteins with specific posttranslational modifications.

Molecular hydrogel-immobilized enzymes exhibit superactivity and high stability in organic solvents.

This communication describes the use of a molecular hydrogel to immobilize enzymes for catalyzing reactions in an organic solvent to attain superactivity and exceptional stability.

The first pamidronate containing polymer and copolymer.

Here we report the synthesis, characterization and hydrogelation of polymers consisting of pamidronate, a useful therapeutic agent.

Biocompatible fluorescent supramolecular nanofibrous hydrogel for long-term cell tracking and tumor imaging applications.

Biocompatible peptide-based supramolecular hydrogel has recently emerged as a new and promising system for biomedical applications. In this work, Rhodamine B is employed as a new capping group of self-assembling peptide, which not only provides the driving force for supramolecular nanofibrous hydrogel formation, but also endows the hydrogel with intrinsic fluroescence signal, allowing for various bioimaging applications. The fluorescent peptide nanofibrous hydrogel can be formed via disulfide bond reduction. After dilution of the hydrogel with aqueous solution, the fluorescent nanofiber suspension can be obtained. The resultant nanofibers are able to be internalized by the cancer cells and effectively track the HeLa cells for as long as 7 passages. Using a tumor-bearing mouse model, it is also demonstrated that the fluorescent supramolecular nanofibers can serve as an efficient probe for tumor imaging in a high-contrast manner.

Using phosphatases to generate self-assembled nanostructures and their applications.

SIGNIFICANCE: Self-assembled nanostructures have received significant research interest in the last decade, because they show great promise for drug delivery, diagnostics, tissue engineering, and regenerative medicine. Recently, the development of enzyme-assisted self-assembled nanostructures has become an active area of research because of the attractive characteristics of enzymes, such as ready availability, good biocompatibility, and high selectivity and specificity. Phosphatases, taking part in approximately 30% of intra- and extracellular activities, have been widely employed as triggers for the generation of self-assembled biomaterials, including static, reversible, and dynamic systems. RECENT ADVANCES: In this review, we highlight the generation of self-assembled systems of synthetic molecules using phosphatases and their potential applications. We first summarize the generation of different kinds of static and dynamic self-assembled structures, including nanofibers and nanoparticles, by the dephosphorylation reaction catalyzed by phosphatases. The antagonistic interactions of phosphatases and kinases make this system one of the most attractive candidates for biotransformation. Diverse biomedical applications of phosphatases/kinases-involved self-assembled systems have been extensively explored in fields such as bacterial growth inhibition, drug delivery, imaging of self-assembly inside live cells, and biomineralization. We then summarize the reversible self-assembled systems controlled by the pair enzymes of phosphatases/kinases, in which different morphologies of self-assembled nanostructures can be achieved and switched by the pair enzymes. These phosphatase-involved self-assembled systems can be used for many applications such as controlled drug delivery, enzyme activity imaging, and cancer cell inhibition. CRITICAL ISSUES: Phosphatases are over-expressed in several cancer cell lines. Their detection is, therefore, important for cancer diagnostics. Nanomaterials that can respond to abnormal phosphatase activities also have big potential for the delivery of therapeutic agents on demand. The study of reversible self-assembling systems control by the phosphatase/kinase switch may provide useful insights to understand the working principle of this important biological switch. FUTURE DIRECTIONS: The design principle mentioned in this review may stimulate the generation of smart self-assembled systems by other enzymes or other pairs of enzymes. The combination of environment-sensitive fluorescence property of fluorescent dyes and self-assembling molecules that can respond to enzymes may lead to the development of smart probes to monitor important biological processes.

Rational design of multifunctional hetero-hexameric proteins for hydrogel formation and controlled delivery of bioactive molecules.

A hetero-hexameric protein system is developed in this study, which not only functions as cross-linkers for hydrogel formation but also offers docking sites for controlled delivery of bioactive molecules. First, a hexameric protein with two, four, and six tax-interacting protein-1 (TIP-1), respectively (named as 2T, 4T, and 6T), is designed and obtained. As the hexapeptide ligand (WRESAI) can specifically bind to TIP-1 with high affinity, the hexameric proteins of 2T, 4T, and 6T can be used to crosslink the self-assembling nanofibers of Nap-GFFYGGGWRESAI, leading to formation of injectable biohybrid hydrogels with tunable mechanical properties. Furthermore, a hetero-hexameric protein containing four TIP-1 and two C-terminal moiety of the pneumococcal cell-wall amidase LytA (C-LytA) proteins is designed and engineered (named as 4T2C). The 4T2C proteins can not only serve as cross-linkers for hydrogel formation but also provide docking sites for loading and controlled release of model drug Rhoda-GGK'. This study opens up new opportunities for further development of multifunctional hetero- recombinant protein-based hydrogels for biological applications.

Glutathione-triggered formation of molecular hydrogels for 3D cell culture.

The development of three dimensional (3D) scaffolds that are suitable for cell encapsulation and proliferation is highly important for tissue engineering and regenerative medicine. We reported in this paper on several molecular hydrogels formed through glutathione (GSH) reduction, whose mechanical property and zeta potential could be regulated by concentration and structure of gelators in resulting gels, respectively. The hydrogels were characterized by several techniques including rheology, TEM and fluorescence. We found that, in our system, the mechanical property of hydrogels but not the zeta potential of self-assembled structures had big influences on mouse fibroblast 3T3 cells spreading and proliferation. Hydrogels with storage modulus (G') of hundreds of pascals (Pa) were suitable for 3T3 cells spreading and proliferation. We believed that hydrogels reported in this study had big potential for applications in different fields, such as 3D cell culture and tissue engineering.

Multifunctional biohybrid hydrogels for cell culture and controlled drug release.

Here we reported a novel biohybrid hydrogel system with the potential for cell culture and controlled drug release.

Enzyme-controllable delivery of nitric oxide from a molecular hydrogel.

A ß-galactosidase-responsive molecular hydrogelator of a nitric oxide (NO) donor can release NO in a controllable manner to improve wound healing.

Janus nanogels of PEGylated Taxol and PLGA-PEG-PLGA copolymer for cancer therapy.

Nanogels are promising carriers for the delivery of anti-cancer drugs for cancer therapy. We report in this study on a Janus nanogel system formed by mixing a prodrug of Taxol (PEGylated Taxol) and a copolymer of PLGA-PEG-PLGA. The Janus nanogels have good stability over months in aqueous solutions and the freeze-dried powder of nanogels can be re-dispersed instantly in aqueous solutions. The Janus nanogels show an enhanced inhibition effect on tumor growth in a mice breast cancer model probably due to the enhanced uptake of the nano-sized materials by the EPR effect. What is more, the nanogels can also serve as physical carriers to co-deliver other anti-cancer drugs such as doxorubicin to further improve the anti-cancer efficacy. The results obtained from H&E staining and TUNEL assay also support the observation of tumor growth inhibition. These results suggest the potential of this novel delivery system for cancer therapy.