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.

Radiant star nanoparticle prodrugs were synthesized in a two-step process by first homopolymerizing RAFT transmers followed by copolymerization from the hyperbranched polymer core.

Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis, can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages, We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection.

Herein, we developed a fully polymerizable, peptide-targeted, camptothecin polymeric prodrug system. Two prodrug monomers were synthesized via esterification of campothecin (20Cam) and 10-hydroxycamptothecin (10Cam) with mono-2-(methacryloyloxy)ethyl succinate (SMA) resulting in polymerizable forms of the aliphatic ester- and aromatic ester-linked drugs respectively. These monomers were then incorporated into zwitterionic polymers via RAFT copolymerization of the prodrug monomers with a tert-butyl ester protected carboxy betaine monomer. Subsequent deprotection of the tert-butyl residues with TFA yielded carboxy betaine methacrylate (CBM) scaffolds with controlled prodrug incorporation. Reverse phase HPLC was then employed to establish drug release kinetics in human serum at 37 oC for the resultant polymeric prodrugs. Copolymers containing 10Cam residues linked via aromatic esters showed faster hydrolysis rates with 59 % drug released at 7 days, while copolymers with Cam residues linked via aliphatic esters showed only 28 % drug release over the same time period. These differences in drug release kinetics were then shown to correlate with large differences in cytotoxic activity in SKOV3 ovarian cancer cell cultures. At 72 hours, the IC50s of aromatic- and aliphatic- ester linked prodrugs were 56 nM and 4776 nM, respectively. An EGFR-targeting peptide sequence, GE11, was then directly incorporated into the polymeric prodrugs via RAFT copolymerization of the polymeric prodrugs with a peptide macronomer. The GE11-targeted polymeric prodrugs showed enhanced targeting and cytotoxic activity in SKOV3 cell cultures relative to untargeted polymers containing the negative control sequence HW12. Following pulse-chase treatment (15 min, 37 °C), the 72 hour IC50 of GE11 targeted prodrug was determined to be 1597 nM, in contrast to 3399 nM for the non-targeted control.

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.

Prodrug monomers derived from the antibiotic ciprofloxacin were synthesized with phenolic or aliphatic esters linking the drug to a polymerizable methacrylate group.

Reversible addition–fragmentation chain transfer (RAFT) polymerization was employed to prepare prodrug polymer carrier systems with the chemotherapeutic agent camptothecin (Cam) and the kinase inhibitor dasatinib (Dt).

Hydrophilic, hydrophobic, and combinations of these monomers were directly (co)polymerized via RAFT polymerization induced self-assembly (PISA) in acetic acid.