Directional Submicrofiber Hydrogel Composite Scaffolds Supporting Neuron Differentiation and Enabling Neurite Alignment

Cell cultures aiming at tissue regeneration benefit from scaffolds with physiologically relevant elastic moduli to optimally trigger cell attachment, proliferation and promote differentiation, guidance and tissue maturation. Complex scaffolds designed with guiding cues can mimic the anisotropic nature of neural tissues, such as spinal cord or brain, and recall the ability of human neural progenitor cells to differentiate and align. This work introduces a cost-efficient gelatin-based submicron patterned hydrogel–fiber composite with tuned stiffness, able to support cell attachment, differentiation and alignment of neurons derived from human progenitor cells. The enzymatically crosslinked gelatin-based hydrogels were generated with stiffnesses from 8 to 80 kPa, onto which poly(ε-caprolactone) (PCL) alignment cues were electrospun such that the fibers had a preferential alignment. The fiber–hydrogel composites with a modulus of about 20 kPa showed the strongest cell attachment and highest cell proliferation, rendering them an ideal differentiation support. Differentiated neurons aligned and bundled their neurites along the aligned PCL filaments, which is unique to this cell type on a fiber–hydrogel composite. This novel scaffold relies on robust and inexpensive technology and is suitable for neural tissue engineering where directional neuron alignment is required, such as in the spinal cord

Synthesis of Zwitterionic, Hydrophobic, and Amphiphilic Polymers: Via RAFT Polymerization Induced Self-Assembly (PISA) in Acetic Acid

Polymerization induced self-assembly (PISA) in acetic acid was employed to polymerize the hydrophilic sulfobetaine monomer 2-(N-3-sulfopropyl-N,N-dimethyl ammonium)ethyl methacrylate (DMAPS) and the hydrophobic monomer lauryl methacrylate (LMA). Polymerizations were conducted from a macro chain transfer agent (macro-CTA) consisting of 66% 2-hydroxyethyl methacrylate (HEMA) and 33% poly(ethylene glycol) methyl ether methacrylate FW ~ 300 Da (O300). A degree of polymerization (DP) of 50 was targeted for the macro-CTA in order to yield diblock copolymers with significantly larger 2nd blocks. From the poly(HEMA-co-O300) macro-CTA, diblock copolymers of poly[(HEMA-co-O300)-b-(DMAPS)] and poly[(HEMA-co-O300)-b-(LMA)] were grown via PISA in acetic acid. In order to maintain colloidal stability, it was necessary to conduct PISA of DMAPS at 10 wt% monomer, while LMA polymerizations maintained stability at 20 wt% monomer. Mnvs. conversion plots for both DMAPS and LMA show linear increases in molecular weight over the course of the polymerizations. Analysis of the molecular weight distributions revealed a progressive narrowing throughout the polymerization from an initial bimodal state. Copolymers of DMAPS and LMA were also synthesized over a large range of comonomer feed ratios. These materials show composition-dependent sizes in buffered solutions between 11 nm for the copolymer containing 80% by mol DMAPS to 75 nm for the copolymer containing 40 mol% DMAPS. PISA in acetic acid was then used to prepare copolymers of DMAPS with a range of hydrophobic polymerizable prodrug monomers as well as a polymerizable peptide macromonomer. The resultant copolymers had narrow molecular weight distributions and were readily soluble in saline solutions

Nanostructured Glycopolymer Augmented Liposomes to Elucidate Carbohydrate-Mediated Targeting

Carbohydrate receptors on alveolar macrophages are attractive targets for receptor-mediated delivery of nanostructured therapeutics. In this study, we employed reversible addition fragmentation chain transfer polymerization to synthesize neoglycopolymers, consisting of mannose- and galactose methacrylate-based monomers copolymerized with cholesterol methacrylate for use in functional liposome studies. Glycopolymer-functional liposomes were employed to elucidate macrophage mannose receptor (CD206) and macrophage galactose-type lectin (CD301) targeting in both primary macrophage and immortal macrophage cell lines. Expression of CD206 and CD301 was observed to vary significantly between cell lines (murine alveolar macrophage, murine bone marrow-derived macrophage, RAW264.7, and MH-S), which has significant implications in in vitro targeting and uptake studies. Synthetic glycopolymers and glycopolymer augmented liposomes demonstrated specific receptor-mediated uptake in a manner dependent on carbohydrate receptor expression. These results establish a platform capable of probing endogenous carbohydrate receptor-mediated targeting via glycofunctional nanomaterials

Macrophage-Targeted Drugamers with Enzyme-Cleavable Linkers Deliver High Intracellular Drug Dosing and Sustained Drug Pharmacokinetics against Alveolar Pulmonary Infections

Intracellular bacterial infections localized to the lung alveolar macrophage (AM) remain one of the most challenging settings for antimicrobial therapy. Current systemic antibiotic treatment fails to deliver sustained doses to intracellular bacterial reservoirs, which necessitates prolonged treatment regimens. Herein, we demonstrate a new intracellular enzyme-cleavable polymeric prodrug with tailored ciprofloxacin release profiles in the lungs and AM. The targeted polymeric prodrug, termed “drugamers”, incorporates (1) hydrophilic mannose residues to solubilize the antibiotic cargo and to target and enhance AM uptake and intracellular delivery, and (2) enzyme-cleavable linkage chemistry to provide high and sustained intracellular AM drug dosing. Prodrug monomers, derived from the antibiotic ciprofloxacin, were synthesized with either an intracellular protease cleavable dipeptide linker or a hydrolytic phenyl ester linker. RAFT polymerization was used to copolymerize the prodrug monomers and mannose monomer to synthesize well-defined drugamers without requiring a post-polymerization conjugation step. In addition to favorable in vivo safety profiles following intratracheal administration, a single dose of the drugamers sustained ciprofloxacin dosing in lungs and AMs above the minimum inhibitory concentration (MIC) over at least a 48 h period. The enzyme-cleavable therapeutic achieved a \u3e 10-fold increase in sustained ciprofloxacin in AM, and maintained a significantly higher whole lung PK as well. Ciprofloxacin dosed in identical fashion displayed rapid clearance with a half-life of approximately 30 min. Notably, inhalation of the mannose-targeted ciprofloxacin drugamers achieved full survival (100%) in a highly lethal mouse model of pneumonic tularemia, contrasted with 0% survival using free ciprofloxacin. These findings demonstrate the versatility of the drugamer platform for engineering the intracellular pharmacokinetic profiles and its strong therapeutic activity in treating pulmonary intracellular infections

Mannose Conjugated Polymer Targeting P. Aeruginosa Biofilms

Biofilms are one of the most challenging obstacles in bacterial infections. By providing protection against immune responses and antibiotic therapies, biofilms enable chronic colonization and the development of antibiotic resistance. As previous clinical observations and studies have shown, traditional antibiotic therapy alone cannot effectively treat and eliminate biofilm forming infections due to the protection conferred by the biofilm. A new strategy specifically targeting biofilms must be developed. Here, we specifically target and bind to the PAO1 biofilm and elucidate the molecular mechanism behind the interaction between a glycan targeted polymer and biofilm using a continuous flow biofilm model. The incubation of biofilms with fluorescent glycan targeted polymers demonstrated strong and persistent interactions with the mannose-containing polymer even after 24 h of continuous flow. To evaluate the role of major biofilm proteins LecB and CdrA, loss of function experiments with knockout variants established the dual involvement of both proteins in mannose targeted polymer retention. These results identify a persistent and specific targeting strategy to the biofilm, emphasizing its potential value as a delivery strategy and encouraging further exploration of biofilm targeted delivery

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy Against Aerosolized Intracellular Alveolar Infections

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating Franciscella tularensis pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16-17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal F.t. novicida mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding Cmax/MIC and AUC0-24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as F. tularensis, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy against Aerosolized Intracellular Alveolar Infections

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating <i>Franciscella tularensis</i> pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16–17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal <i>F.t. novicida</i> mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding <i>C</i>_max/MIC and AUC_0–24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as <i>F. tularensis</i>, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles