Engineering of an environmentally responsive beta roll peptide for use as a calcium-dependent cross-linking domain for peptide hydrogel formation.

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.

Polymeric Nanocarriers With Mucus-Diffusive and Mucus-Adhesive Properties to Control Pharmacokinetic Behavior of Orally Dosed Cyclosporine A.

The present study develops cyclosporine A (CsA)-loaded polymeric nanocarriers with mucus-diffusive and mucus-adhesive potential to control pharmacokinetic behavior after oral administration for the treatment of inflammatory bowel diseases (IBD). CsA-loaded nanocarriers consisting of polystyrene-block-polyethylene glycol (PEG-CsA) and polystyrene-block-polyacrylic acid (PAA-CsA) were prepared by a flash nanoprecipitation. Both nanocarriers showed redispersibility from lyophilized powder back to uniform nanocarrier with a mean diameter of approximately 150 nm. The nanocarriers exhibited significantly improved release behavior of CsA under pH 6.8 condition compared. A test of mucodiffusion, using artificial mucus, demonstrated the mucus-diffusive and mucus-adhesive potential of PEG-CsA and PAA-CsA, respectively, dependent on the lack of electrostatic interactions between the surface-coated polymer and mucin. Oral administrations of PEG-CsA and PAA-CsA (10 mg-CsA/kg) in rats resulted in significant improvements of absorption, as evidenced by 50- and 25-fold higher bioavailability than crude CsA, respectively. PAA-CsA exhibited more sustained and slower absorption process of CsA than PEG-CsA because of the different diffusion behavior within the mucus layer. In the rat model of IBD, significant suppression of inflammatory symptoms could be achieved by oral treatment with both CsA nanoparticles. These polymeric nanocarriers are promising dosage options to control pharmacokinetic behavior of orally dosed CsA, contributing to the development of safe and effective treatment for IBD.

Pseudomonas aeruginosa pyocyanin production reduced by quorum-sensing inhibiting nanocarriers.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes a wide range of infections; it is becoming increasingly difficult to treat due to antibiotic resistance. Quorum-sensing (QS) based therapeutics, which function by disabling pathogen virulence without killing pathogens, are a promising class of drugs that may be used to treat bacterial infections without eliciting resistance development. The use of QS drugs to treat pulmonary P. aeruginosa infections, however, has been greatly limited due to the inability to deliver QS drugs at sufficiently high concentrations past physiological barriers such as pulmonary mucus. Here we apply a block copolymer-directed self-assembly process, Flash NanoPrecipitation, to develop a series of QS-active formulations that are fully water dispersible, stable, and mucus-penetrating. These formulations inhibit P. aeruginosa virulence without inhibiting cell growth. Particle size (70 nm-400 nm) and release rate (1 h-14 days) can be tuned by altering constructs' physical properties and formulation excipients. We also demonstrate, to the best of our knowledge, the first instance of a QS nanocarrier platform technology that can penetrate through human cystic fibrosis pulmonary mucus. This work highlights the need to incorporate nanoformulation strategies into the development of next-generation antimicrobial therapeutics.

Narrow Absorption NIR Wavelength Organic Nanoparticles Enable Multiplexed Photoacoustic Imaging.

Photoacoustic (PA) imaging is an emerging hybrid optical-ultrasound based imaging technique that can be used to visualize optical absorbers in deep tissue. Free organic dyes can be used as PA contrast agents to concurrently provide additional physiological and molecular information during imaging, but their use in vivo is generally limited by rapid renal clearance for soluble dyes and by the difficulty of delivery for hydrophobic dyes. We here report the use of the block copolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to form series of highly hydrophobic optical dyes into stable, biocompatible, and water-dispersible nanoparticles (NPs) with sizes from 38 to 88 nm and with polyethylene glycol (PEG) surface coatings suitable for in vivo use. The incorporation of dyes with absorption profiles within the infrared range, that is optimal for PA imaging, produces the PA activity of the particles. The hydrophobicity of the dyes allows their sequestration in the NP cores, so that they do not interfere with targeting, and high loadings of >75 wt % dye are achieved. The optical extinction coefficients (ε (mL mg(-1) cm(-1))) were essentially invariant to the loading of the dye in NP core. Co-encapsulation of dye with vitamin E or polystyrene demonstrates the ability to simultaneously image and deliver a second agent. The PEG chains on the NP surface were functionalized with folate to demonstrate folate-dependent targeting. The spectral separation of different dyes among different sets of particles enables multiplexed imaging, such as the simultaneous imaging of two sets of particles within the same animal. We provide the first demonstration of this capability with PA imaging, by simultaneously imaging nontargeted and folate-targeted nanoparticles within the same animal. These results highlight Flash NanoPrecipitation as a platform to develop photoacoustic tools with new diagnostic capabilities.

Injectable shear-thinning hydrogels engineered with a self-assembling Dock-and-Lock mechanism.

Injected therapeutics, such as cells or biological molecules, may have enhanced efficiency when delivered within a scaffold carrier. Here, we describe a dual-component Dock-and-Lock (DnL) self-assembly mechanism that can be used to construct shear-thinning, self-healing, and injectable hydrogels. One component is derived from the RIIα subunit of cAMP-dependent kinase A and is engineered as a telechelic protein with end groups that dimerize (docking step). The second component is derived from the anchoring domain of A-kinase anchoring protein (AD) and is attached to multi-arm crosslinker polymers and binds to the docked proteins (locking step). When mixed, these two DnL components form robust physical hydrogels instantaneously and under physiological conditions. Mechanical properties and erosion rates of DnL gels can be tuned through the AD peptide sequence, the concentration and ratio of each component, and the number of peptides on the cross-linking polymer. DnL gels immediately self-recover after deformation, are resistant to yield at strains as high as 400%, and completely self-heal irrespective of prior mechanical disruption. Mesenchymal stem cells mixed in DnL gels and injected through a fine needle remain highly viable (>90%) during the encapsulation and delivery process, and encapsulated large molecules are released with profiles that correspond to gel erosion. Thus, we have used molecular engineering strategies to develop cytocompatible and injectable hydrogels that have the potential to support cell and drug therapies.