A novel microporous biomaterial vaccine platform for long-lasting antibody mediated immunity against viral infection.

Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude, neutralization, and duration of anti-receptor binding domain antibodies compared to Alum vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.

A novel microporous biomaterial vaccine platform for long-lasting antibody mediated immunity against viral infection.

Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4+ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude and duration of anti-receptor binding domain antibodies compared to Alum and mRNA-vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.

Resistance to complement activation, cell membrane hypersialylation and relapses in chronic lymphocytic leukemia patients treated with rituximab and chemotherapy.

The anti-CD20-specific monoclonal antibody rituximab (RTX), in combination with chemotherapy, is commonly used for primary treatment in chronic lymphocytic leukemia (CLL). However, relapses remain important and activation of the complement pathway is one of the mechanisms by which RTX generates the destruction of B cells directly by complement-dependent cytotoxicity (CDC), or indirectly by antibody-dependent cellular phagocytosis. In this study, the RTX capacity to induce CDC was established in 69 untreated CLL patients, this cohort including 34 patients tested before the initiation of RTX-chemotherapy. In vitro CDC-resistance to RTX predicts lower response rates to RTX-chemotherapy and shorter treatment free survival. Furthermore, the predictive value of CDC-resistance was independent from the clinical, cytogenetic and FcγR3A V158F polymorphism status. In contrast, CLL cell resistance to CDC predominates in IGHV unmutated patients and was related to an important α2-6 sialyl transferase activity, which in turn increases cell surface α2-6 hypersialylation. Suspected factors associated with resistance to CDC (CD20, CD55, CD59, factor H, GM1, and sphingomyelin) were not differentially expressed or recruited between the two CLL groups. Altogether, results provide evidence that testing RTX capacity to induce CDC in vitro represents an independent predictive factor of therapeutic effects of RTX, and that α2-6 hypersialylation in CLL cells controls RTX response through the control of the complement pathway. At a time when CLL therapy is moving towards chemo-free treatments, further experiments are required to determine whether performing an initial in vitro assay to appreciate CLL CDC resistance might be useful to select patients.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or   l-tryptophan were selectively recognized by previously identified dopamine or l-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though, slightly greater than the previously determined solution dissociation constant. Using prefunctionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with l-3,4-dihydroxyphenylalanine, l- threo-dihydroxyphenylserine, and l-5-hydroxytryptophan enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons and future identification and characterization of novel aptamers targeting neurotransmitters or other important small molecules.

Small-Molecule Patterning via Prefunctionalized Alkanethiols.

Interactions between small molecules and biomolecules are important physiologically and for biosensing, diagnostic, and therapeutic applications. To investigate these interactions, small molecules can be tethered to substrates through standard coupling chemistries. While convenient, these approaches co-opt one or more of the few small-molecule functional groups needed for biorecognition. Moreover, for multiplexing, individual probes require different surface functionalization chemistries, conditions, and/or protection/deprotection strategies. Thus, when placing multiple small-molecules on surfaces, orthogonal chemistries are needed that preserve all functional groups and are sequentially compatible. Here, we approach high-fidelity small-molecule patterning by coupling small-molecule neurotransmitter precursors, as examples, to monodisperse asymmetric oligo(ethylene glycol)alkanethiols during synthesis and prior to self-assembly on Au substrates. We use chemical lift-off lithography to singly and doubly pattern substrates. Selective antibody recognition of pre-functionalized thiols was comparable to or better than recognition of small molecules functionalized to alkanethiols after surface assembly. These findings demonstrate that synthesis and patterning approaches that circumvent sequential surface conjugation chemistries enable biomolecule recognition and afford gateways to multiplexed small-molecule functionalized substrates.

Designing Hybrid Antibiotic Peptide Conjugates To Cross Bacterial Membranes.

We design hybrid antibiotic peptide conjugates that can permeate membranes. Integration of multiple components with different functions into a single molecule is often problematic, due to competing chemical requirements for different functions and to mutual interference. By examining the structure of antimicrobial peptides (AMPs), we show that it is possible to design and synthesize membrane active antibiotic peptide conjugates (MAAPCs) that synergistically combine multiple forms of antimicrobial activity, resulting in unusually strong activity against persistent bacterial strains.

Pentobra: A Potent Antibiotic with Multiple Layers of Selective Antimicrobial Mechanisms against Propionibacterium Acnes.

Although antibiotics are a common treatment for acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in the skin are compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule that has multiple mechanisms of antibacterial action and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides. Pentobra demonstrated potent and selective killing of P. acnes but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes clinical strains. Moreover, electron microscopic studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing antibiotics against P. acnes that are effective in cutaneous environments.

Synthetic and bioinspired cage nanoparticles for drug delivery.

Nanotechnology has the potential to revolutionize drug delivery, but still faces some limitations. One of the main issues regarding conventional nanoparticles is their poor drug-loading and their early burst release. Thus, to overcome these problems, researchers have taken advantage of the host-guest interactions that drive some assemblies to form cage molecules able to strongly entrap their cargo and design new nanocarriers called cage nanoparticles. These systems can be classified into two categories: bioinspired nanosystems such as virus-like particles, ferritin, small heat shock protein: and synthetic host-guest supramolecular systems that require engineering to actually form supramolecular nanoassemblies. This review will highlight the recent advances in cage nanoparticles for drug delivery with a particular focus on their biomedical applications.

Engineering persister-specific antibiotics with synergistic antimicrobial functions.

Most antibiotics target growth processes and are ineffective against persister bacterial cells, which tolerate antibiotics due to their reduced metabolic activity. These persisters act as a genetic reservoir for resistant mutants and constitute a root cause of antibiotic resistance, a worldwide problem in human health. We re-engineer antibiotics specifically for persisters using tobramycin, an aminoglycoside antibiotic that targets bacterial ribosomes but is ineffective against persisters with low metabolic and cellular transport activity. By giving tobramycin the ability to induce nanoscopic negative Gaussian membrane curvature via addition of 12 amino acids, we transform tobramycin itself into a transporter sequence. The resulting molecule spontaneously permeates membranes, retains the high antibiotic activity of aminoglycosides, kills E. coli and S. aureus persisters 4-6 logs better than tobramycin, but remains noncytotoxic to eukaryotes. These results suggest a promising paradigm to renovate traditional antibiotics.

Phenylboronic acid-installed polymeric micelles for targeting sialylated epitopes in solid tumors.

Ligand-mediated targeting of nanocarriers to tumors is an attractive strategy for increasing the efficiency of chemotherapies. Sialylated glycans represent a propitious target as they are broadly overexpressed in tumor cells. Because phenylboronic acid (PBA) can selectively recognize sialic acid (SA), herein, we developed PBA-installed micellar nanocarriers incorporating the parent complex of the anticancer drug oxaliplatin, for targeting sialylated epitopes overexpressed on cancer cells. Following PBA-installation, the micelles showed high affinity for SA, as confirmed by fluorescence spectroscopy even at intratumoral pH conditions, i.e., pH 6.5, improving their cellular recognition and uptake and enhancing their in vitro cytotoxicity against B16F10 murine melanoma cells. In vivo, PBA-installed micelles effectively reduced the growth rate of both orthotopic and lung metastasis models of melanoma, suggesting the potential of PBA-installed nanocarriers for enhanced tumor targeting.

"Click" conjugation of peptide on the surface of polymeric nanoparticles for targeting tumor angiogenesis.

PURPOSE: Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site. METHODS: The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as "click" reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both "click" coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line. RESULTS: Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained. CONCLUSIONS: These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional platform for cancer treatment if coupled with therapeutic agents.