André J. van der Vlies

In situ cell manipulation through enzymatic hydrogel photopatterning

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)--a key ECM crosslinking enzyme--is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose

Significance High adjuvant doses are generally required to induce strong CD8+ T-cell immunity with subunit vaccines. Here we codeliver an antigen and an adjuvant coupled on separate ultrasmall polymeric nanoparticles. Because both payloads are attached to similarly sized nanoparticles, and as size is the principle determinant of nanoparticle drainage, this enhanced the dual uptake of antigen and adjuvant by cross-presenting dendritic cells resident in the draining lymph nodes. This cotargeting induced potent effector CD8+ T cells and a more powerful memory recall of these cytotoxic T cells compared with nanoparticle-conjugated antigen with free adjuvant. As such, nanoparticle conjugation enhanced the immunogenicity of adjuvants while maintaining a low dose, and thus limiting toxicity, affecting the design of future subunit vaccine formulations. In subunit vaccines, strong CD8+ T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8+ T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 μg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

Carbon Monoxide-Releasing Micelles for Immunotherapy

With the discovery of important biological roles of carbon monoxide (CO), the use of this gas as a therapeutic agent has attracted attention. However, the medical application of this gas has been hampered by the complexity of the administration method. To overcome this problem, several transition-metal carbonyl complexes, such as Ru(CO)(3)Cl(glycinate), [Ru(CO)(3)Cl(2)](2), and Fe(η(4)-2-pyrone)(CO)(3), have been used as CO-releasing molecules both in vitro and in vivo. We sought to develop micellar forms of metal carbonyl complexes that would display slowed diffusion in tissues and thus better ability to target distal tissue drainage sites. Specifically, we aimed to develop a new CO-delivery system using a polymeric micelle having a Ru(CO)(3)Cl(amino acidate) structure as a CO-releasing segment. The CO-releasing micelles were prepared from triblock copolymers composed of a hydrophilic poly(ethylene glycol) block, a poly(ornithine acrylamide) block bearing Ru(CO)(3)Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) block. The polymers formed spherical micelles in the range of 30-40 nm in hydrodynamic diameter. Further characterization revealed the high CO-loading capacity of the micelles. CO-release studies showed that the micelles were stable in physiological buffer and serum and released CO in response to thiol-containing compounds such as cysteine. The CO release of the micelles was slower than that of Ru(CO)(3)Cl(glycinate). In addition, the CO-releasing micelles efficiently attenuated the lipopolysaccharide-induced NF-κB activation of human monocytes, while Ru(CO)(3)Cl(glycinate) did not show any beneficial effects. Moreover, cell viability assays revealed that the micelles significantly reduced the cytotoxicity of the Ru(CO)(3)Cl(amino acidate) moiety. This novel CO-delivery system based on CO-releasing micelles may be useful for therapeutic applications of CO.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8+ T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8+ T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4+ T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8+ T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8+ T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Design and synthesis of polymeric hydrogen sulfide donors.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that has several important biological functions in the human body. Because of the difficulties of handling H2S gas, small organic compounds that release H2S under physiological conditions have been developed. The observed bioactivities of these H2S donors have generally been directly correlated with their H2S release properties. However, apart from H2S release, these H2S donors also exert biological effects by direct interaction with intracellular components within the cytoplasm after passive diffusion across cellular membranes. Here we report polymeric H2S donors based on ADT-OH which would alter cellular trafficking of ADT-OH to minimize the unfavorable interactions with intracellular components. We designed and synthesized a poly(ethylene glycol)-ADT (PEG-ADT) conjugate having ADT linked via an ether bond. Whereas ADT-OH significantly reduced cell viability in murine macrophages, the PEG-ADT conjugate did not show obvious cytotoxicity. The PEG-ADT conjugate released H2S in murine macrophages but not in the presence of serum proteins. The PEG-ADT conjugate was taken up by the cell through the endocytic pathway and stayed inside endolysosomes, which is different from the small amphiphilic donor ADT-OH that can directly enter the cytoplasm. Furthermore, PEG-ADT was capable of potentiating LPS-induced inflammation. This polymeric H2S donor approach may help to better understand the H2S bioactivities of the H2S donor ADT-OH.

Preparation of Well-Defined Ibuprofen Prodrug Micelles by RAFT Polymerization

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat acute pain, fever, and inflammation and are being explored in a new indication in cancer. Side effects associated with long-term use of NSAIDs such as gastrointestinal damage and elevated risk of stroke, however, can limit their use and exploration in new indications. Here we report a facile method to prepare well-defined amphiphilic diblock copolymer NSAID prodrugs by direct reversible addition-fragmentation transfer (RAFT) polymerization of the acrylamide derivative of ibuprofen (IBU), a widely used NSAID. The synthesis and self-assembling behavior of amphiphilic diblock copolymers (PEG-PIBU) having a hydrophilic poly(ethylene glycol) block and a hydrophobic IBU-bearing prodrug block were investigated. Release profiles of IBU from the micelles by hydrolysis were evaluated. Furthermore, the antiproliferative action of the IBU-containing micelles in human cervical carcinoma (HeLa) and murine melanoma (B16-F10) cells was assessed.

Inhibition of angiogenesis by antioxidant micelles.

Antioxidant micelles capable of scavenging reactive oxygen species (ROS) are prepared from poly(ethylene glycol)-b-poly(dopamine) block copolymers. The micelles inhibit tube formation of human umbilical vein endothelial cells (HUVECs) by scavenging endogenous ROS. Furthermore, the micelles inhibit angiogenesis in the chicken ex ovo chorioallantoic membrane assay. The results show that antioxidant micelles containing catechol moieties may be useful in anti-angiogenic therapy to treat various diseases such as cancer.

Polymeric micelles for hydrogen sulfide delivery

Polymeric micelles for therapeutic delivery of hydrogen sulfide (H2S) were developed. The micelles released H2S in murine macrophages and enhanced proinflammatory responses induced by a toll-like receptor 7 ligand, gardiquimod. This micellar H2S delivery system may have potential in immunotherapy and vaccine development.

Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis

Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column.

Preparation of poly(γ-glutamic acid)/hydroxyapatite monolith via biomineralization for bone tissue engineering

A hybrid monolith of poly(γ-glutamic acid) and hydroxyapatite (PGA/HAp monolith) was prepared via biomineralization and used as a macroporous cell scaffold in bone tissue engineering. The PGA monolith having a bimodal pore size distribution was used as a substrate to induce biomineralization. The PGA/HAp monolith was obtained by immersing the PGA monolith in simulated body fluid. Pretreatment with CaCl2 enhanced the apatite-forming ability of the PGA monolith. Murine osteoblastic MC3T3-E1 cells efficiently attached and proliferated on the PGA/HAp monolith. MTT assay showed that both the PGA and PGA/HAp monolith did not have apparent cytotoxicity. Moreover, the PGA and PGA/HAp monoliths adsorbed bone morphogenetic protein-2 (BMP-2) by electrostatic interaction which was slowly released in the medium during cell culture. The PGA/HAp monolith enhanced BMP-2 induced alkaline phosphatase activity compared to the PGA monolith and a polystyrene culture plate. Thus, these PGA/HAp monoliths may have potential in bone tissue engineering.