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The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. At present, only two oral preexposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, because it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long-acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous (SC) delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, because of potentially limited efficacy and possible toxicity issues with SC delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

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.

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.

Pulmonary melioidosis is a bacterial disease with high morbidity and a mortality rate that can be as high as 40% in resource-poor regions of South Asia. This disease burden is linked to the pathogen's intrinsic antibiotic-resistance and protected intracellular localization in alveolar macrophages. Current treatment regimens require several antibiotics with multi-month oral and intravenous administrations that are difficult to implement in the developing world. Herein, we report that a macrophage-targeted polyciprofloxacin prodrug acts as a surprisingly effective pre-exposure prophylactic in highly lethal murine models of aerosolized human pulmonary melioidosis. A single dose of the polymeric prodrug maintained high lung drug levels and targeted an intracellular depot of ciprofloxacin to the alveolar macrophage compartment that was sustained over a period of 7 days above minimal inhibitory concentrations. This intracellular pharmacokinetic profile provided complete pre-exposure protection in a BSL-3 model with a drug-resistant, aerosolized clinical isolate of Burkholderia pseudomallei from Thailand. This total protection was achieved despite the bacteria's intrinsic resistance to ciprofloxacin and where an equivalent dose of pulmonary-administered ciprofloxacin was ineffective. For the first time, we demonstrate that targeting the intracellular macrophage compartment with extended antibiotic dosing can achieve pre-exposure prophylaxis in a model of pulmonary melioidosis. This fully synthetic and modular therapeutic platform could be an important therapeutic approach with new or re-purposed antibiotics for melioidosis prevention and treatment, especially as portable inhalation devices in high-risk, resource-poor settings.

Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.

Clinical studies have validated that antiretroviral (ARV) drugs can serve as an HIV pre-exposure prophylactic (PrEP) strategy. Dosing adherence remains a crucial factor determining the final efficacy outcomes, and both long-acting implants and injectable depot systems are being developed to improve patient adherence. Here, we describe an injectable depot platform that exploits a new mechanism for both formation and controlled release. The depot is a polymeric prodrug synthesized from monomers that incorporate an ARV drug tenofovir alafenamide (TAF) with degradable linkers that can be designed to control release rates. The prodrug monomers are synthetically incorporated into homopolymer or block designs that exhibit high drug weight percent (wt%) and also are hydrophobized in these prodrug segments to drive depot formation upon injection. Drug release converts those monomers to more hydrophilic pendant groups via linker cleavage, and as this drug release proceeds, the polymer chains losing hydrophobicity are then disassociated from the depot and released over time to provide a depot dissolution mechanism. We show that long-acting TAF depots can be designed as block copolymers or as homopolymers. They can also be designed with different linkers, for example with faster or slower degrading p-hydroxybenzyloxycarbonyl (Benzyl) and ethyloxycarbonyl (Alkyl) linkers, respectively. Diblock designs of p(glycerol monomethacrylate)-b-p(Alkyl-TAF-methacrylate) and p(glycerol monomethacrylate)-b-p(Benzyl-TAF-methacrylate) were first characterized in a mouse subcutaneous injection model. The alkylcarbamate linker design (TAF 51 wt%) showed excellent sustained release profiles of the key metabolite tenofovir (TFV) in skin and plasma over a 50-day period. Next, the homopolymer design with a high TAF drug wt% of 73% was characterized in the same model. The homopolymer depots with p(Alkyl-TAFMA) exhibited sustained TFV and TAF release profiles in skin and blood over 60 days, and TFV-DP concentrations in peripheral blood mononuclear cells (PBMC) were found to be at least 10-fold higher than the clinically suggested minimally EC90 protective concentration of 24 fmol/106 cells. These are the first reports of sustained parent TAF dosing observed in mouse and TFV-DP in mouse PBMC. IVIS imaging of rhodamine labeled homopolymer depots showed that degradation and release of the depot coincided with the sustained TAF release. Finally, these polymers showed excellent stability in accelerated stability studies over a six-month time period, and exceptional solubility of over 700 mg/mL in the DMSO formulation solvent. The homopolymer designs have a drug reservoir potential of well over a year at mg/day dosing and may not require cold chain storage for global health and developed world long-acting drug delivery applications.

HIV and hepatitis B are two of the most prevalent viruses globally, and despite readily available preventive treatments unforgiving treatment regimens still exist, such as daily doses of medicine that are challenging to maintain especially in poorer countries. More advanced and longer-lasting delivery vehicles can potentially overcome this problem by reducing maintenance requirements and significantly increase access to medicine. Here, we designed a technology to control the delivery of an antiviral drug over a long timeframe via a nanofiber based delivery scaffold that is both easy to produce and use. An antiviral prodrug containing tenofovir alafenamide (TAF) was synthesized by initial conjugation to glycerol monomethacrylate followed by polymerization to form a diblock copolymer (pTAF) using reversible addition-fragmentation chain transfer (RAFT). In order to generate an efficient drug delivery system this copolymer was fabricated into an electrospun nanofiber (ESF) scaffold using blend electrospinning with poly(caprolactone) (PCL) as the carrier polymer. SEM images revealed that the pTAF-PCL ESFs were uniform with an average diameter of (787 ± 0.212 nm), while XPS analysis demonstrated that the pTAF was overrepresented at the surface of the ESFs. Additionally, the pTAF exhibited a sustained release profile over a 2 month period in human serum (HS), suggesting that these types of copolymer-based drugamers can be used in conjunction with electrospinning to produce long-lasting drug delivery systems.

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 greater than 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.

Radiant star nanoparticle (RSN) prodrugs were synthesized in a two-step process by first homopolymerizing RAFT transmers followed by copolymerization from the hyperbranched polymer core. Two trithiocarbonate-based transmers were synthesized containing either alkyl ester or acetal groups linking the polymerizable methacrylate group to the chain transfer agent (CTA). RAFT polymerization from the homopolymerized transmer cores yielded RSNs with linear polymer chains connected to hyperbranched cores. Hydrolysis studies conducted over a period of 30 days at 37 °C in acetate buffer showed that RSNs prepared from alkyl ester linked cores remained stable while acetal linked cores exhibited a progressive degradation into linear polymers over the same period. Macrophage targeting RSN prodrugs containing the antibiotic ciprofloxacin and receptor-targeting mannose residues were synthesized directly via RAFT polymerization of the prodrug and mannose monomers. Hydrolysis studies conducted in human serum showed that the RSNs released the covalently linked ciprofloxacin significantly faster than diblock copolymer micelles but moderately slower than soluble copolymers with comparable compositions. Flow cytometry showed substantially higher macrophage binding by the mannose-targeted RSNs while in vivo biocompatibility experiments showed no differences relative to phosphate buffer treated negative controls.

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 (o)C 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.

Engineered immune cells are an exciting therapeutic modality which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering has been developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed “drugamers”. The fluorescein ligand facilitated stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporated prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustained anti-proliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system was demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporated a small molecule trans-gene activator, CMP8, that switched-on a degron-tagged gene circuit to provide temporal regulation of engineered T cell protein expression. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both anti-tumor and transgene-activating small molecule prodrugs.

The complex tumor microenvironment (TME) of PDA creates a uniquely immune- and drug-privileged sanctuary that contributes to disease pathogenesis and treatment resistance. The profoundly immunosuppressive microenvironment of PDA includes abundant MDSC, Treg, M2-like TAM and a dearth of cytotoxic T-cells. Recent discoveries of highly expressed Toll-like receptors 7 and 8 (TLR7/8) in the desmoplastic stroma of PDA suggest that TLR7/8 agonists may be an effective immune-targeted therapy. TLR7/8 agonists may inhibit tumor growth by any of several mechanisms including myeloid cell reeducation, depletion of MDSC, and/or depletion of Treg, thereby activating cytotoxic CD8+ T cells. However, the circulatory half-life of most TLR7/8 agonists is relatively short, and elevated systemic levels and prolonged treatment can also cause severe adverse reactions that can limit their use and effectiveness. To address these concerns, we have developed a novel polymeric form of a TLR7/8 agonist that exploits a breakthrough synthetic technology to directly polymerize drugs into nanocarriers with defined architectures. These agents are designed to minimize adverse effects and increase efficacy through more favorable pharmacokinetics and enhanced delivery to desmoplastic tumors with release profiles tailorable over the range of days to weeks. This agent is in development in our murine preclinical trials program (MCTP) which is modeled on human clinical trials and involves randomized, blinded, placebo-controlled studies against the current standard-of-care using the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48Cre/+ (KPC) model as our primary platform of the autochthonous disease. Exploratory experiments were performed to determine optimal dose and treatment schedule. KPC mice with ultrasound-imaging documented disease meeting enrollment criteria were subsequently randomized into one of two arms in 28-day pilot studies: control vehicle-treated and polymeric TLR7/8 (n=5-8 animals per arm). Daily health and behavior checks, serial body weight measurements, and complete blood count (CBC) profiles over the course of the study revealed no adverse health effects or overt toxicities. Mice were euthanized at study completion and all organs harvested for histological analyses; single cell suspensions from blood, spleen, peri-pancreatic lymph nodes and primary tumor were analyzed by FACS and/or CyTOF Helios cytometry (incorporating 30+ specific markers and with a particular focus on identifying specific states and subtypes of CD8+ T cells as well as markers of checkpoint activation to assess innate and adaptive immunity in treated versus untreated and tumor versus normal tissues). Preliminary analyses reveal response associated with increases in T cell activation markers and concomitant changes in inhibitory markers. M1 and M2 macrophage populations also appeared to be profoundly affected, suggesting targeting of specific immune cell types and distinct activation states. Citation Format: Christopher C. DuFort, Ciana L. Lopez, Martin C. Whittle, Vladimir Vlaskin, Aditi Vadodkar, Selvi Srinivasan, Patrick S. Stayton, Sunil R. Hingorani. Overcoming stromal barriers in PDA with a novel polymeric Toll-like receptor agonist [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-105.

Peptide-based cancer vaccines offer production and safety advantages but have had limited clinical success due to their intrinsic instability, rapid clearance, and low cellular uptake. Nanoparticle-based delivery vehicles can improve the in vivo stability and cellular uptake of peptide antigens. Here, a well-defined, self-assembling mannosylated polymer is developed for anti-cancer peptide antigen delivery. The amphiphilic polymer is prepared by RAFT polymerization, and the peptide antigens are conjugated to the pH-sensitive hydrophobic block through the reversible disulfide linkage for selective release after cell entry. The polymer-peptide conjugates self-assemble into sub-100 nm micelles at physiological pH and dissociate at endosomal pH. The mannosylated micellar corona increases the accumulation of vaccine cargoes in the draining inguinal lymph nodes and facilitates nanoparticle uptake by professional antigen presenting cells. In vivo studies demonstrate that the mannosylated micelle formulation improved dendritic cell activation and enhanced antigen-specific T cell responses, resulting in higher antitumor immunity in tumor-bearing mice compared to free peptide antigen. The mannosylated polymer is therefore a simple and promising platform for the delivery of peptide cancer vaccines. This article is protected by copyright. All rights reserved.

Peptides derived from the third Bcl-2 homology domain (BH3) renormalize apoptotic signaling by antagonizing prosurvival Bcl-2 family members. These potential peptide drugs exhibit therapeutic activities but are limited by barriers including short circulation half-lives and poor penetration into cells. A diblock polymeric micelle carrier for the BIM BH3 peptide was recently described that demonstrated antitumor activity in a B-cell lymphoma xenograft model [Berguig et al., Mol. Ther. 2015, 23, 907–917]. However, the disulfide linkage used to conjugate the BIM peptide was shown to have nonoptimal blood stability. Here we describe a peptide macromonomer composed of BIM capped with a four amino acid cathepsin B substrate (FKFL) that possesses high blood stability and is cleaved to release the drug inside of target cells. Employing RAFT polymerization, the peptide macromonomer was directly integrated into a multifunctional diblock copolymer tailored for peptide delivery. The first polymer block was made as a macro-chain transfer agent (CTA) and composed of a pH-responsive endosomolytic formulation of N,N-diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA). The second polymer block was a copolymer of the peptide and polyethylene glycol methacrylate (PEGMA). PEGMA monomers of two sizes were investigated (300 Da and 950 Da). Protein gel analysis, high performance liquid chromatography, and coupled mass spectrometry (MS) showed that incubation with cathepsin B specifically cleaved the FKFL linker and released active BIM peptide with PEGMA300 but not with PEGMA950. MALDI-TOF MS showed that incubation of the peptide monomers alone in human serum resulted in partial cleavage at the FKFL linker after 12 h. However, formulation of the peptides into polymers protected against serum-mediated peptide degradation. Dynamic light scattering (DLS) demonstrated pH-dependent micelle disassembly (25 nm polymer micelles at pH 7.4 versus 6 nm unimers at pH 6.6), and a red blood cell lysis assay showed a corresponding increase in membrane destabilizing activity (

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads® in terms of biomarker isolation kinetics, assay speed and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab’s binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (K(d)), ~ 2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by 10’s of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.

In order to improve the detection limit of existing HIV diagnostic assays, we explored the use of a temperature-responsive magnetic nanoparticle reagent system in conjunction with cyanovirin-N for HIV recognition to rapidly and efficiently concentrate viral particles from larger sample volumes, ~ 1 ml. Cyanovirin-N (CVN) mutant, Q62C, was expressed, biotinylated, and then complexed with a thermally responsive polymer-streptavidin conjugate. Confirmation of protein expression/activity was performed using matrix assisted laser desorption/ionization (MALDI) and a TZM-bl HIV inhibition assay. Biotinylated CVN mutant recognition with gp120 was characterized using surface plasmon resonance (SPR). Virus separation and enrichment using a thermoresponsive magnetic nanoparticle reagent system were measured using RT-PCR. Biotinylated Q62C exhibited a KD of 0.6 nM to gp120. The temperature-responsive binary reagent system achieved a maximum viral capture of nearly 100% HIV, 1 × 105 virus copies in 100 μl, using pNIPAAm-Q62C within 30 minutes. Additionally, the same reagent system achieved nearly 9-fold enrichment by processing a 10-times larger sample of 1000 μl (Fig. 3). This work demonstrated a temperature-responsive reagent system that provides enrichment of HIV using antiviral lectin CVN for recognition, which is potentially amenable for use in point-of-care settings.

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). Copolymers were prepared as dense polyethylene glycol brushes via direct copolymerization of the prodrug macromonomers with polyethylene glycol methacrylate (O950, FW ∼ 950 daltons). The brushes display controlled drug release profiles with little burst or late-phase release aberrations. Hydrolysis studies of the hydrophilic copolymers conducted in human serum showed 33 ± 1.7 and 22 ± 2.4% drug release over the course of 144 h for the ester linked Dt and Cam respectively. Polymer morphology was also shown to play a key role in drug release rates. Copolymers with the drug distributed in the copolymer segment showed faster release rates than diblock copolymers where the hydrophobic drug molecules were localized in discreet hydrophobic blocks. The latter materials were shown to self-assemble into polymeric micelles with the drug block separated from the aqueous phase. Live animal imaging in PC-3 (human prostate cancer cell line) tumor xenographs showed that the fluorescently labeled copolymer brushes were trafficked to the tumor 24 hours post injection. Ex vivo analysis of the harvested tissues showed that polymer accumulated in the tumor with kidney excretion. In vitro cytotoxicity measurements conducted in K562-S and K562-R cells demonstrated ability of the macromolecular conjugates to release active drugs. The direct copolymerization of different drug classes into controlled copolymers via RAFT, together with their favorable release profiles, suggest these carriers merit further study as therapeutic systems.

Prodrug monomers derived from the antibiotic ciprofloxacin were synthesized with phenolic or aliphatic esters linking the drug to a polymerizable methacrylate group. RAFT polymerization of these monomers exhibited linear pseudo-first-order kinetics and Mnvs. conversion plots, and low Đ values throughout the polymerization. Prodrug monomers were then copolymerized with polyethyleneglycol methacrylate to yield hydrophilic copolymers with narrow Đ values. A poly(O950) macroCTA was also synthesized and chain extended with the antibiotic monomers to form diblock copolymers. The resultant copolymers and diblock copolymers were characterized with 1H and 19F NMR and found to contain 16.5 and 30–35 wt% ciprofloxacin, respectively. DLS measurements demonstrated that the copolymers remained unimeric between pH 5.6–7.4, while the diblock copolymers formed nanoparticles with diameters between 30–40 nm at physiological pH. Drug release kinetics were measured in human serum via HPLC analysis. Copolymers containing ciprofloxacin linked via phenolic esters showed faster hydrolysis rates with 50% drug released at 120 h, whereas copolymers with the corresponding aliphatic ester linkages showed the same drug release over 22 d. Diblock copolymers with a discrete ciprofloxacin block and a poly(O950) stabilizing block self-assembled into micelles, and exhibited reduced hydrolysis rates for both ester linked drugs. In vitro toxicity measurements in RAW 264.7 cells showed the copolymers to be nontoxic up to 20 mg mL−1 following a 24 h incubation period. The polymer drugs were shown to be active against Burkholderia thailandensis in a bacteria-macrophage co-culture model of melioidosis with MIC values of 6.0 and 0.6 mM for the aliphatic and phenyl ester linked copolymeric prodrugs, respectively.

Small magnetic nanoparticles that have surfaces decorated with stimuli-responsive polymers can be reversibly aggregated via a stimulus, such as temperature, to enable efficient and rapid biomarker separation. To fully realize the potential of these particles, the synthesis needs to be highly reproducible and scalable to large quantity. We have developed a new synthesis for temperature-responsive magnetic nanoparticles via an in situ coprecipitation process of Fe(2+)/Fe(3+) salts at room temperature with poly(acrylic acid)-block-poly(N-isopropylacrylamide) diblock copolymer template, synthesized via the reversible addition− fragmentation chain-transfer polymerization method. These particles were 56% polymer by weight with a 6.5:1 Fe/COOH ratio and demonstrated remarkable stability over a 2 month period. The hydrodynamic diameter remained constant at ~28 nm with a consistent transition temperature of 34 °C, and the magnetic particle separation efficiency at 40 °C was ≥95% over the 2 month span. These properties were maintained for all large-scale synthesis batches. To demonstrate the practical utility of the stimuli-responsive magnetic nanoparticles, the particles were incorporated into a temperature-responsive binary reagent system and efficiently separated a model protein biomarker (mouse IgG) as well as purified extracellular vesicles derived from a human biofluid, seminal plasma. The ease of using these particles will prove beneficial for various biomedical applications.

Aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to prepare a series of linear copolymers of N,N-dimethylacrylamide (DMA) and 2-hydroxyethylacrylamide (HEAm) with narrow Đ values over a molecular weight range spanning three orders of magnitude (103 to 106 Da). Trithiocarbonate-based RAFT chain transfer agents (CTAs) were grafted onto these scaffolds using carbodiimide chemistry catalyzed with DMAP. The resultant graft chain transfer agent (gCTA) was subsequently employed to synthesize polymeric brushes with a number of important vinyl monomer classes including acrylamido, methacrylamido, and methacrylate. Brush polymerization kinetics were evaluated for the aqueous RAFT polymerization of DMA from a 10 arm gCTA. Polymeric brushes containing hydroxyl functionality were further functionalized in order to prepare 2nd generation gCTAs which were subsequently employed to prepare polymers with a brushed-brush architecture with molecular weights in excess of 106 Da. These resultant single particle nanoparticles (SNPs) were employed as drug delivery vehicles for the anthracycline-based drug doxorubicin via copolymerization of DMA with a protected carbazate monomer (bocSMA). Cell-specific targeting functionality was also introduced via copolymerization with a biotin-functional monomer (bioHEMA). Drug release of the hydrazone linked doxorubicin was evaluated as function of pH and serum and chemotherapeutic activity was evaluated in SKOV3 ovarian cancer cells.

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 C

A thienopyrrole oligomer conjoined to DNA was prepared by means of a templated synthesis protocol. The oligomer was formed by reaction, initiated with HRP/H2O2, of thieno[3,2-b]pyrrole monomers attached to cytosine bases. The thienopyrrole oligomer was characterized spectroscopically.

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.

Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody–drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody–drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines.

Adsorption isotherms, circular dichroism (CD) spectroscopy, x-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to investigate the adsorption of human osteocalcin (hOC) and decarboxylated (i.e., Gla converted back to Glu) hOC (dhOC) onto various calcium phosphate surfaces as well as silica surfaces. The adsorption isotherms and XPS nitrogen signals were used to track the amount of adsorbed hOC and dhOC. The intensities of key ToF-SIMS amino acid fragments were used to assess changes in the structure of adsorbed hOC and dhOC. CD spectra were used to investigate the secondary structure of OC. The largest differences were observed when the proteins were adsorbed onto silica versus calcium phosphate surfaces. Similar amounts (3-4 at. % N) of hOC and dhOC were adsorbed onto the silica surface. Higher amounts of hOC and dhOC were adsorbed on all the calcium phosphate surfaces. The ToF-SIMS data showed that the intensity of the Cys amino acid fragment, normalized to intensity of all amino acid fragments, was significantly higher (∼×10) when the proteins were adsorbed onto silica. Since in the native OC structure the cysteines are located in the center of three α-helices, this indicates both hOC and dhOC are more denatured on the silica surface. As hOC and dhOC denature upon adsorption to the silica surface, the cysteines become more exposed and are more readily detected by ToF-SIMS. No significant differences were detected between hOC and dhOC adsorbed onto the silica surface, but small differences were observed between hOC and dhOC adsorbed onto the calcium phosphate surfaces. In the OC structure, the α-3 helix is located above the α-1 and α-2 helices. Small differences in the ToF-SIMS intensities from amino acid fragments characteristic of each helical unit (Asn for α-1; His for α-2; and Phe for α-3) suggests either slight changes in the orientation or a slight uncovering of the α-1 and α-2 for adsorbed dhOC. XPS showed that similar amounts of hOC and dhOC were absorbed onto hydroxyapaptite and octacalcium phosphate surfaces, but ToF-SIMS detected some small differences in the amino acid fragment intensities on these surfaces for adsorbed hOC and dhOC.

Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to prepare a series of copolymers consisting of 2-hydroxyethyl methacrylate (HEMA) and poly(ethylene glycol) methyl ether methacrylate (FWavg ∼ 950 Da) (O950) with variable comonomer compositions and molecular weights for use as polymeric scaffolds. Reactivity ratios for the monomer pair were determined to be 1.37 and 0.290 respectively. To these scaffolds trithiocarbonate-based RAFT chain transfer agents (CTAs) were grafted using carbodiimide chemistry. The resultant graft chain transfer agents (gCTA) were subsequently employed to polymerize dimethylaminoethyl methacrylate (DMAEMA) and (HPMA) between degrees of polymerization (DP) of 25 and 200. Kinetic analysis for the polymerization of DMAEMA targeting a DP of 100 from a 34 arm graft gCTA show linear Mn conversion and pseudo first order rate plots with narrow molecular weight distributions that move toward lower elution volumes with monomer conversion. Đ values for these polymerizations remain low at around 1.20 at monomer conversions as high as 70%. pH-responsive endosomalytic brushes capable of spontaneously self-assembling into polymersomes were synthesized and a combination of dynamic light scattering (DLS), cryoTEM, and red blood cell hemolysis were employed to evaluate the aqueous solution properties of the polymeric brush as a function of pH. Successful encapsulation of ceftazidime and pH-dependent drug release properties were confirmed by HPLC. Intracellular antibiotic activity of the drug-loaded polymersomes was confirmed in a macrophage coculture model of infection with B. thailandensis and RAW 264.7 cells.

Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, the development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and the use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior. (1, 2) These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system because the folate receptor is an attractive target for tumor-selective therapies as a result of its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer.