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Post-operative complications of vascular anastomosis procedures remain a significant clinical challenge and health burden globally. Each year, millions of anastomosis procedures connect arteries and/or veins in vascular bypass, vascular access, organ transplant, and reconstructive surgeries, generally via suturing. Dysfunction of these anastomoses, primarily due to neointimal hyperplasia and the resulting narrowing of the vessel lumen, results in failure rates of up to 50% and billions of dollars in costs to the healthcare system. Non-absorbable sutures are the gold standard for vessel anastomosis; however, damage from the surgical procedure and closure itself causes an inflammatory cascade that leads to neointimal hyperplasia at the anastomosis site. Here, we demonstrate the development of a novel, scalable manufacturing system for fabrication of high strength sutures with nanofiber-based coatings composed of generally regarded as safe (GRAS) polymers and either sirolimus, tacrolimus, everolimus, or pimecrolimus. These sutures provided sufficient tensile strength for maintenance of the vascular anastomosis and sustained drug delivery at the site of the anastomosis. Tacrolimus-eluting sutures provided a significant reduction in neointimal hyperplasia in rats over a period of 14 days with similar vessel endothelialization in comparison to conventional nylon sutures. In contrast, systemically delivered tacrolimus caused significant weight loss and mortality due to toxicity. Thus, drug-eluting sutures provide a promising platform to improve the outcomes of vascular interventions without modifying the clinical workflow and without the risks associated with systemic drug delivery.

The unique cancer-associated immunosuppression in brain, combined with a paucity of infiltrating T cells, contributes to the low response rate and poor treatment outcomes of T cell-based immunotherapy for patients diagnosed with glioblastoma multiforme (GBM). Here, we report on a self-assembling paclitaxel (PTX) filament (PF) hydrogel that stimulates macrophage-mediated immune response for local treatment of recurrent glioblastoma. Our results suggest that aqueous PF solutions containing aCD47 can be directly deposited into the tumor resection cavity, enabling seamless hydrogel filling of the cavity and long-term release of both therapeutics. The PTX PFs elicit an immune-stimulating tumor microenvironment (TME) and thus sensitizes tumor to the aCD47-mediated blockade of the antiphagocytic “don’t eat me” signal, which subsequently promotes tumor cell phagocytosis by macrophages and also triggers an antitumor T cell response. As adjuvant therapy after surgery, this aCD47/PF supramolecular hydrogel effectively suppresses primary brain tumor recurrence and prolongs overall survivals with minimal off-target side effects.

Purpose: Prior clinical trials evaluating cisplatin for non–muscle-invasive bladder cancer (NMIBC) were stopped due to local and systemic toxicity. Currently, there is still a need for improved intravesical therapies, and nanoparticle-based CDDP may be efficacious without the toxicity of free cisplatin observed in the past.Experimental Design: Cisplatin nanoparticles (CDDP NPs) were developed using biocompatible poly(l-aspartic acid sodium salt; PAA), both with and without low and high grafting density of methoxy-polyethylene glycol (PEG). In vitro cytotoxicity studies confirmed activity of CDDP NPs and CDDP solution against a papillary bladder cancer cell line. Local toxicity was assessed by three weekly intravesical administrations of CDDP formulations. CDDP NPs and CDDP solution were evaluated for bladder absorption in murine models 1 and 4 hours after intravesical administration. In vivo efficacy was evaluated in an immunocompetent carcinogen model of NMIBC.Results: CDDP NPs showed decreased local toxicity, as assessed by bladder weight, compared with CDDP solution. Furthermore, >2 μg/mL of platinum was observed in mouse serum after intravesical administration of CDDP solution, whereas serum platinum was below the limit of quantification after intravesical administration of CDDP NPs. CDDP NPs provided significantly increased (P < 0.05) drug levels in murine bladders compared with CDDP solution for at least 4 hours after intravesical administration. In vivo, CDDP NPs reduced cancer cell proliferation compared with untreated controls, and was the only treatment group without evidence of invasive carcinoma.Conclusions: Cisplatin-loaded PAA NPs have the potential to improve intravesical treatment of NMIBC while reducing local and systemic side effects. Clin Cancer Res; 23(21); 6592–601. ©2017 AACR.

Supplementary Table 1: Nanoparticle physicochemical characteristics. Table S2: IC50 values of various formulations against various bladder cancer cell lines.

Supplementary Figure 1: Transmission electron micrographs of PAA-CDDP NPs, PEGlow-PAA-CDDP NPs and PEGhigh-PAA-CDDP NPs (Scale bars = 500 nm). Supplementary Figure 2. Apoptosis was compared in RT4 cells treated with CDDP and PAA-CDDP NPs. DNA (blue, stained with hoechst33342), apoptotic cells (green, stained with Apoxin), and dead cells (red, stained with propidium iodide). Both formulations induced apoptosis in RT4 cells. Supplementary Figure 3: Images of gross tissue size supported the increased bladder weights in the CDDP treated group after 3 weekly administrations compared to PAA-CDDP NP, PEGlow-PAA-CDDP NPs and untreated control. Supplementary Figure 4. (A) Representative IHC sections stained for cleaved caspase 3 (brown) of rats treated with intravesical CDDP solution or intravesical PAA-CDDP NPs. (B) No significant differences in caspase 3+ cells were identified between treatment groups.

Topical corticosteroid eye drop is the mainstay for preventing and treating corneal graft rejection. While the frequent topical corticosteroid use is associated with risk of intraocular pressure (IOP) elevation and poor patient compliance that leads to graft failure and the requirement for a repeated, high-risk corneal transplantation. Here, we developed dexamethasone sodium phosphate (DSP)–loaded dicarboxyl-terminated poly(lactic acid) nanoparticle (PLA DSP-NP) formulations with relatively high drug loading (8 to 10 weight %) and 6 months of sustained intraocular DSP delivery in rats with a single dosing. PLA DSP-NP successfully reversed early signs of corneal rejection, leading to rat corneal graft survival for at least 6 months. Efficacious PLA DSP-NP doses did not affect IOP and showed no signs of ocular toxicity in rats for up to 6 months. Subconjunctival injection of DSP-NP is a promising approach for safely preventing and treating corneal graft rejection with the potential for improved patient adherence.

IntroductionInhaled gene therapy of muco-obstructive lung diseases requires a strategy to achieve therapeutically relevant gene transfer to airway epithelium covered by particularly dehydrated and condensed mucus gel layer. Here, we introduce a synthetic DNA-loaded mucus-penetrating particle (DNA-MPP) capable of providing safe, widespread and robust transgene expression in in vivo and in vitro models of muco-obstructive lung diseases.MethodsWe investigated the ability of DNA-MPP to mediate reporter and/or therapeutic transgene expression in lung airways of a transgenic mouse model of muco-obstructive lung diseases (ie, Scnn1b-Tg) and in air–liquid interface cultures of primary human bronchial epithelial cells harvested from an individual with cystic fibrosis. A plasmid designed to silence epithelial sodium channel (ENaC) hyperactivity, which causes airway surface dehydration and mucus stasis, was intratracheally administered via DNA-MPP to evaluate therapeutic effects in vivo with or without pretreatment with hypertonic saline, a clinically used mucus-rehydrating agent.ResultsDNA-MPP exhibited marked greater reporter transgene expression compared with a mucus-impermeable formulation in in vivo and in vitro models of muco-obstructive lung diseases. DNA-MPP carrying ENaC-silencing plasmids provided efficient downregulation of ENaC and reduction of mucus burden in the lungs of Scnn1b-Tg mice, and synergistic impacts on both gene transfer efficacy and therapeutic effects were achieved when DNA-MPP was adjuvanted with hypertonic saline.DiscussionDNA-MPP constitutes one of the rare gene delivery systems providing therapeutically meaningful gene transfer efficacy in highly relevant in vivo and in vitro models of muco-obstructive lung diseases due to its unique ability to efficiently penetrate airway mucus.

While eye drops are the most common ocular dosage form, eye drops for treating diseases of the posterior segment (retina, choroid, optic nerve) have yet to be developed. In glaucoma, eye drops are used extensively for delivering intraocular pressure (IOP) lowering medications to the anterior segment. However, degeneration of retinal ganglion cells (RGCs) in the retina may progress despite significant IOP lowering, suggesting that a complementary neuroprotective therapy would improve glaucoma management. Here, we describe a hypotonic, thermosensitive gel-forming eye drop for effective delivery of sunitinib, a protein kinase inhibitor with activity against the neuroprotective targets dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK), to enhance survival of RGCs after optic nerve injury. Further, binding of sunitinib to melanin in the pigmented cells in the choroid and retinal pigment epithelium (RPE) led to prolonged intraocular residence time, including therapeutically relevant concentrations in the non-pigmented retinal tissue where the RGCs reside. The combination of enhanced intraocular absorption provided by the gel-forming eye drop vehicle and the intrinsic melanin binding properties of sunitinib led to significant protection of RGCs with only once weekly eye drop dosing. For a chronic disease such as glaucoma, an effective once weekly eye drop for neuroprotection could result in greater patient adherence, and thus, greater disease management and improved patient quality of life.

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma., In asthma, mucus plugging is an important cause of airflow obstruction, but it is not targeted by widely used bronchodilator and anti-inflammatory drugs. Here the authors show that reduction of disulfide bonds that hold mucin polymers together reverses asthma-like obstruction in mice.

Artificial vitreous humor holds immense potential for use in in vitro intravitreal drug delivery assays. In this study, we investigated rheological properties and drug or nanoparticle migration in hyaluronic acid (HA) – agar based hydrogels and compared these characteristics with bovine vitreous humor. Gel compositions identified in literature containing HA (0.7–5.0 mg/ml) and agar (0.95–4.0 mg/ml) were classified as either high (VH), medium (VM) or low (VL) polymer load. Viscoelastic behavior was evaluated using oscillatory rheology, and migration of differently sized and charged polystyrene nanoparticles (NPs) through the different gels was determined via multiple particle tracking. Comparable rheological behaviour was observed between VL and bovine vitreous. Tracking evaluations revealed that increasing particle size and gel viscosity slowed NP migration. Additionally, 100 nm anionic NPs migrated slower than neutral NPs in VL and VM, while cationic NPs were immobile in all gels. Finally, distribution and clearance of sodium fluorescein was used to model drug mobility through the gels using a custom-built eye model. Flow and angular movement only influenced drug migration in VL and VM, but not VH. Finally, VL and VM demonstrated to have the most similar sodium fluorescein clearance to that of bovine vitreous humor. Together, these evaluations demonstrate that low viscosity HA-agar gels can be used to approximate nanoparticle and drug migration through biological vitreous humor.

Noninfectious uveitis is a potentially blinding ocular condition that often requires treatment with corticosteroids to prevent inflammation-related ocular complications. Severe forms of uveitis such as panuveitis that affects the whole eye often require a combination of topical and either regional or systemic corticosteroid. Regional corticosteroids are currently delivered inside the eye by intravitreal injection (e.g. Ozurdex®, an intravitreal dexamethasone implant). Intravitreal injection is associated with rare but potentially serious side effects, including endophthalmitis, retinal and vitreous hemorrhage, and retinal detachment. Subconjunctival (SCT) injection is a less invasive option that is a common route used for post-surgical drug administration and treatment of infection and severe inflammation. However, it is the water soluble form of dexamethasone, dexamethasone sodium phosphate (DSP), that has been demonstrated to achieve high intraocular penetration with subconjunctival injection. It is difficult to load highly water soluble drugs, such as DSP, and achieve sustained drug release using conventional encapsulation methods. We found that use of carboxyl-terminated poly(lactic-co-glycolic acid) (PLGA) allowed encapsulation of DSP into biodegradable nanoparticles (NP) with relatively high drug content (6% w/w) if divalent zinc ions were used as an ionic “bridge” between the PLGA and DSP. DSP-Zn-NP had an average diameter of 210 nm, narrow particle size distribution (polydispersity index ~0.1), and near neutral surface charge (−9 mV). DSP-Zn-NP administered by SCT injection provided detectable DSP levels in both the anterior chamber and vitreous chamber of the eye for at least 3 weeks. In a rat model of experimental autoimmune uveitis (EAU), inflammation was significantly reduced in both the front and back of the eye in animals that received a single SCT injection of DSP-Zn-NP as compared to animals that received either aqueous DSP solution or phosphate buffered saline (PBS). DSP-Zn-NP efficacy was evidenced by a reduced clinical disease score, decreased expression of various inflammatory cytokines, and preserved retinal structure and function. Furthermore, SCT DSP-Zn-NP significantly reduced microglia cell density in the retina, a hallmark of EAU in rats. DSP-Zn-NP hold promise as a new strategy to treat noninfectious uveitis and potentially other ocular inflammatory disorders.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.

Sutures are applied almost universally at the site of trauma or surgery, making them an ideal platform to modulate the local, postoperative biological response, and improve surgical outcomes. To date, the only globally marketed drug‐eluting sutures are coated with triclosan for antibacterial application in general surgery. Loading drug directly into the suture rather than coating the surface offers the potential to provide drug delivery functionality to microsurgical sutures and achieve sustained drug delivery without increasing suture thickness. However, conventional methods for drug incorporation directly into the suture adversely affect breaking strength. Thus, there are no market offerings for drug‐eluting sutures, drug‐coated, or otherwise, in ophthalmology, where very thin sutures are required. Sutures themselves help facilitate bacterial infection, and antibiotic eye drops are commonly prescribed to prevent infection after ocular surgeries. An antibiotic‐eluting suture may prevent bacterial colonization of sutures and preclude patient compliance issues with eye drops. We report twisting of hundreds of individual drug‐loaded, electrospun nanofibers into a single, ultra‐thin, multifilament suture capable of meeting both size and strength requirements for microsurgical ocular procedures. Nanofiber‐based polycaprolactone sutures demonstrated no loss in strength with loading of 8% levofloxacin, unlike monofilament sutures which lost more than 50% strength. Moreover, nanofiber‐based sutures retained strength with loading of a broad range of drugs, provided antibiotic delivery for 30 days in rat eyes, and prevented ocular infection in a rat model of bacterial keratitis.

Intravesical chemotherapy is a key approach for treating refractory non-muscle-invasive bladder cancer (NMIBC). However, the effectiveness of intravesical chemotherapy is limited by bladder tissue penetration and retention. Here, we describe the development of a docetaxel nanosuspension that, when paired with a low osmolality (hypotonic) vehicle, demonstrates increased uptake by the bladder urothelium with minimal systemic exposure. We compare the bladder residence time and efficacy in an immune-competent rat model of NMIBC to the clinical comparator, solubilized docetaxel (generic Taxotere) diluted for intravesical administration. We found that only the intravesical docetaxel nanosuspension significantly decreased cell proliferation compared to untreated tumor tissues. The results presented here suggest that the combination of nanoparticle-based chemotherapy and a hypotonic vehicle can provide more efficacious local drug delivery to bladder tissue for improved treatment of refractory NMIBC.

The polymeric controlled-delivery vaccination systems were developed with the concept of using a polymer purely as a matrix to achieve a desired release profile, with no direct effect of the polymer on the immune system desired. Research into this emerging field has concentrated on a variety of strategies for producing adjuvant-active polymeric microparticles. The characteristics of the bioerodible microparticle-delivery systems make them promising candidates for use as efficacious vaccines, as well as excellent tools for probing the effects of antigen-release patterns and vaccine formulation parameters on the immune response. Although the ultimate goal of this research is to produce a viable vaccine, the results also provide further insight into the complex mechanisms of adjuvanticity. The development of adjuvant-active microparticulate vaccine-delivery systems seeks to combine the versatility in release profiles obtained with microparticulate-release systems with the additional adjuvant effect sometimes necessary for an effective immune response.

Nanoparticle-based thymulin gene therapy provides near-complete resolution of key pathology of chronic allergic asthma., Despite long-standing efforts to enhance care for chronic asthma, symptomatic treatments remain the only option to manage this highly prevalent and debilitating disease. We demonstrate that key pathology of allergic asthma can be almost completely resolved in a therapeutic manner by inhaled gene therapy. After the disease was fully and stably established, we treated mice intratracheally with a single dose of thymulin-expressing plasmids delivered via nanoparticles engineered to have a unique ability to penetrate the airway mucus barrier. Twenty days after the treatment, we found that all key pathologic features found in the asthmatic lung, including chronic inflammation, pulmonary fibrosis, and mechanical dysregulation, were normalized. We conducted tissue- and cell-based analyses to confirm that the therapeutic intervention was mediated comprehensively by anti-inflammatory and antifibrotic effects of the therapy. We believe that our findings open a new avenue for clinical development of therapeutically effective gene therapy for chronic asthma.

Inflammation contributes to nearly 4 million global premature births annually. Here, we used a mouse model of intrauterine inflammation to test clinically used formulations, as well as engineered nanoformulations, for the prevention of preterm birth (PTB). We observed that neither systemic 17a-hydroxyprogesterone caproate (Makena) nor vaginal progesterone gel (Crinone) was sufficient to prevent inflammation-induced PTB, consistent with recent clinical trial failures. However, we found that vaginal delivery of mucoinert nanosuspensions of histone deacetylase (HDAC) inhibitors, in some cases with the addition of progesterone, prevented PTB and resulted in delivery of live pups exhibiting neurotypical development. In human myometrial cells in vitro, the P4/HDAC inhibitor combination both inhibited cell contractility and promoted the anti-inflammatory action of P4 by increasing progesterone receptor B stability. Here, we demonstrate the use of vaginally delivered drugs to prevent intrauterine inflammation-induced PTB resulting in the birth of live offspring in a preclinical animal model.

Several generations of poly(β-amino ester) (PBAE) polymers have been developed for efficient cellular transfection. However, PBAE-based gene vectors, similar to other cationic materials, cannot readily provide widespread gene transfer in the brain due to adhesive interactions with the extracellular matrix (ECM). We thus engineered eight vector candidates using previously identified lead PBAE polymer variants but endowed them with non-adhesive surface coatings to facilitate their spread through brain ECM. Specifically, we screened for the ability to provide widespread gene transfer in tumor spheroids and healthy mouse brains. We then confirmed that a lead formulation provided widespread transgene expression in orthotopically established brain tumor models with an excellent in vivo safety profile. Lastly, we developed a method to store it long-term while fully retaining its brain-penetrating property. This new platform provides a broad utility in evaluating novel genetic targets for gene therapy of brain tumors and neurological disorders in preclinical and clinical settings. Graphical abstract We engineered biodegradable DNA-loaded brain-penetrating nanoparticles (DNA-BPN) possessing small particle diameters ( 70 nm) and non-adhesive surface coatings to facilitate their spread through brain tumor extracellular matrix (ECM). These DNA-BPN provide widespread gene transfer in models recapitulating the ECM barrier, including three-dimensional multicellular tumor spheroids and mice with orthotopically established brain tumor.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in zebrafish and mouse RP models, reasoning drugs effective across species may translate better clinically. We first performed a large-scale phenotypic drug screen using a larval zebrafish model of inducible RP. 2,934 compounds, mostly human-approved drugs, were tested across six concentrations. Statistically, 113 compounds achieved “hit” status. Secondary tests of 42 high-priority hits confirmed eleven lead compounds. Nine leads were then evaluated in mouse RP models, with six exhibiting neuroprotective effects. An analysis of potential mechanisms of action suggested complementary activities. Paired lead compound assays in zebrafish showed additive neuroprotective effects for the majority. These results highlight the value of cross-species phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for patients with RP and IRDs.

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

Intravitreal mobility of nanocarriers may have implications on the efficacy of the encapsulated drug in the treatment of vitreo-retinal diseases, with any changes in the integrity of the vitreous microstructure influencing nanoparticle biodistribution. This study investigated enzymatically digested vitreous models to mimic the aging eye. Collagenase, hyaluronidase, or trypsin was employed to selectively digest the structural components of the vitreous. Physical properties of digested bovine vitreous were initially assessed via texture analysis and oscillatory shear testing. Morphological changes in bovine vitreous microstructure were visualized by scanning electron microscopy and diffusion dynamics of hyaluronic acid coated nanoparticles through degraded porcine and bovine vitreous were examined using fluorescence spectroscopy and multiple particle tracking microscopy, respectively. After enzymatic treatment, the vitreous liquefied and its dynamic mechanical properties significantly changed with a decrease in stiffness and an increase in damping capacity. Micrographs confirmed specific digestion of each of the structural vitreous components. Furthermore, enzymatic degradation reduced steric hindrance and enhanced convective flow within the vitreous, resulting in increased intravitreal nanoparticle mobility which could alter the drug pharmacokinetics.

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.

Pulmonary administration of anti-cytokine antibodies offers a targeted therapy in asthma. However, the rapid elimination of proteins from the lungs limits the efficacy of inhaled medications. PEGylation has been shown to increase the residence time of anti-interleukin (IL)-17A and anti-IL-13 antibody fragments in the lungs and to improve their therapeutic efficacy. Yet, little is known about the factors that affect the residence time of PEGylated antibody fragments in the lungs following pulmonary delivery. In this study, we showed that the molecular weight of polyethylene glycol (PEG), 20kDa or 40kDa, had a moderate effect on the residence time of an anti-IL-17A Fab' fragment in the lungs of mice. By contrast, the site of delivery of the anti-IL-17A and anti-IL-13 Fab' fragments within the lungs had a major impact on their residence time, with the deeper the delivery, the more prolonged the residence time. The nature of the Fab' fragment had an influence on its residence time as well and the anti-IL-17A Fab' benefited more from PEGylation than the anti-IL-13 Fab' did. Acute lung inflammation slightly shortened the residence time of the anti-IL-17A and anti-IL-13 Fab' fragments in the lungs but PEGylation was able to prolong their presence in both the healthy and inflamed lungs. Antibody fragments were predominately located within the airway lumen rather than the lung parenchyma. Transport experiments on monolayers of Calu-3 cells and studies of fluorescence recovery after photobleaching in respiratory mucus showed that mechanisms involved in the prolonged presence of PEGylated Fab' in the airway lumen might include binding to the mucus, reduced uptake by respiratory cells and reduced transport across lung epithelia. Finally, using I125-labeled anti-IL-17A Fab', we showed that the protein fragment hardly penetrated into the lungs following subcutaneous injection, as opposed to pulmonary delivery.