Brain-Penetrating Peptide Shuttles across the Blood-Brain Barrier and Extracellular-like Space.

Systemic delivery of therapeutics into the brain is greatly impaired by multiple biological barriers─the blood-brain barrier (BBB) and the extracellular matrix (ECM) of the extracellular space. To address this problem, we developed a combinatorial approach to identify peptides that can shuttle and transport across both barriers. A cysteine-constrained heptapeptide M13 phage display library was iteratively panned against an established BBB model for three rounds to select for peptides that can transport across the barrier. Using next-generation DNA sequencing and in silico analysis, we identified peptides that were selectively enriched from successive rounds of panning for functional validation in vitro and in vivo. Select peptide-presenting phages exhibited efficient shuttling across the in vitro BBB model. Two clones, Pep-3 and Pep-9, exhibited higher specificity and efficiency of transcytosis than controls. We confirmed that peptides Pep-3 and Pep-9 demonstrated better diffusive transport through the extracellular matrix than gold standard nona-arginine and clinically trialed angiopep-2 peptides. In in vivo studies, we demonstrated that systemically administered Pep-3 and Pep-9 peptide-presenting phages penetrate the BBB and distribute into the brain parenchyma. In addition, free peptides Pep-3 and Pep-9 achieved higher accumulation in the brain than free angiopep-2 and may exhibit brain targeting. In summary, these in vitro and in vivo studies highlight that combinatorial phage display with a designed selection strategy can identify peptides as promising carriers, which are able to overcome the multiple biological barriers of the brain and shuttle different-sized molecules from small fluorophores to large macromolecules for improved delivery into the brain.

Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles.

Although mRNA lipid nanoparticles (LNPs) are highly effective as vaccines, their efficacy for pulmonary delivery has not yet fully been established. A major barrier to this therapeutic goal is their instability during aerosolization for local delivery. This imparts a shear force that degrades the mRNA cargo and therefore reduces cell transfection. In addition to remaining stable upon aerosolization, mRNA LNPs must also possess the aerodynamic properties to achieve deposition in clinically relevant areas of the lungs. We addressed these challenges by formulating mRNA LNPs with SM-102, the clinically approved ionizable lipid in the Spikevax COVID-19 vaccine. Our lead candidate, B-1, had the highest mRNA expression in both a physiologically relevant air-liquid interface (ALI) human lung cell model and in healthy mice lungs upon aerosolization. Further, B-1 showed selective transfection in vivo of lung epithelial cells compared to immune cells and endothelial cells. These results show that the formulation can target therapeutically relevant cells in pulmonary diseases such as cystic fibrosis. Morphological studies of B-1 revealed differences in the surface structure compared to LNPs with lower transfection efficiency. Importantly, the formulation maintained critical aerodynamic properties in simulated human airways upon next generation impaction. Finally, structure-function analysis of SM-102 revealed that small changes in the number of carbons can improve upon mRNA delivery in ALI human lung cells. Overall, our study expands the application of SM-102 and its analogs to aerosolized pulmonary delivery and identifies a potent lead candidate for future therapeutically active mRNA therapies.

Using bugs as drugs: Administration of bacteria-related microbes to fight cancer.

Bioengineering of bacteria-related microbes has demonstrated a great potential in targeted cancer therapy. Presently, the major administration routes of bacteria-related microbes for cancer treatment include intravenous injection, intratumoral injection, intraperitoneal injection, and oral delivery. Routes of bacteria administration are critical since different delivery approaches might exert anticancer effects through diverse mechanisms. Herein, we provide an overview of the primary routes of bacteria administration as well as their advantages and limitations. Furthermore, we discuss that microencapsulation can overcome some of the associated challenges with the administration of free bacteria. We also review the latest advancements in combining functional particles with engineered bacteria to fight cancer, which can be coupled with conventional therapies to improve therapeutic effects. Moreover, we highlight the application prospect of emerging 3D bioprinting in cancer bacteriotherapy, which represents a new paradigm for personalized cancer treatment. Eventually, we provide insights into regulatory expectations and concerns regarding this field for the future translation from bench to clinic.

Manufacturing Stable Bacteriophage Powders by Including Buffer System in Formulations and Using Thin Film Freeze-drying Technology.

PURPOSE: Bacteriophage (phage) therapy has re-gained attention lately given the ever-increasing prevalence of multi-drug resistance 'super-bugs'. To develop therapeutic phage into clinically usable drug products, the strategy of solidifying phage formulations has been implemented to diversify the dosage forms and to overcome the storage condition limitations for liquid phage formulations. METHOD: In our work, we hypothesize and tested that an advanced technology, thin film freeze-drying (TFFD), can be used to produce phage containing dry powders without significantly losing phage viability. Here we selected T7 phage as our model phage in a preliminary screening study. RESULTS: We found that a binary excipient matrix of sucrose and leucine at ratios of 90:10 or 75:25 by weight, protected phage from the stresses encountered during the TFFD process. In addition, we confirmed that incorporating a buffer system in the formulation significantly improved the survival of phage during the initial freezing step and subsequent sublimation step in the solidifying processes. The titer loss of phage in SM buffer (Tris/NaCl/MgSO4) containing formulation was as low as 0.19 log plaque forming units, which indicated that phage function was well preserved after the TFFD process. The presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicated that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating a variety of topical drug delivery routes including pulmonary delivery through dry powder inhalers, nebulization after reconstitution, and intranasal or wound therapy, etc. CONCLUSION: From these findings, we show that introducing buffer system can stabilize phage during dehydration processes, and TFFD, as a novel particle engineering method, can successfully produce phage containing powders that possess the desired properties for bioactivity and potentially for inhalation therapy.

Aerosolizable siRNA-encapsulated solid lipid nanoparticles prepared by thin-film freeze-drying for potential pulmonary delivery.

Lipid nanoparticles are increasingly used for drug and gene delivery, including the delivery of small interfering RNA (siRNA). Pulmonary delivery of drug molecules carried by lipid nanoparticles directly into the lung may improve the treatment of certain lung diseases. The present study was designed to test the feasibility of engineering aerosolizable dry powder of lipid nanoparticles by thin-film freeze-drying (TFFD). Solid lipid nanoparticles (SLNs) comprised of lecithin, cholesterol, and a lipid-polyethylene glycol conjugate were prepared by solvent evaporation. Dry powders of the SLNs were prepared by TFFD, spray drying, or conventional shelf freeze-drying. The physical and aerosol properties of the dry powders as well as the physical properties of the SLNs reconstituted from the dry powders were evaluated. The particle size, polydispersity index, and the zeta potential of the SLNs were preserved after they were subjected to TFFD and reconstitution, but not after they were subjected to conventional shelf freeze-drying and reconstitution, and the dry powder prepared by TFFD showed better aerosol performance properties than that prepared by spray drying. SLNs encapsulated with siRNA can also be successfully transformed into aerosolizable dry powder by TFFD, and subjecting the siRNA-encapsulated SLNs to TFFD did not negatively affect the function of the siRNA. It is concluded that TFFD represents a promising method to prepare aerosolizable dry powder of lipid nanoparticles.

Aerosolizable Lipid Nanoparticles for Pulmonary Delivery of mRNA through Design of Experiments.

Messenger RNA is a class of promising nucleic acid therapeutics to treat a variety of diseases, including genetic diseases. The development of a stable and efficacious mRNA pulmonary delivery system would enable high therapeutic concentrations locally in the lungs to improve efficacy and limit potential toxicities. In this study, we employed a Design of Experiments (DOE) strategy to screen a library of lipid nanoparticle compositions to identify formulations possessing high potency both before and after aerosolization. Lipid nanoparticles (LNPs) showed stable physicochemical properties for at least 14 days of storage at 4 °C, and most formulations exhibited high encapsulation efficiencies greater than 80%. Generally, upon nebulization, LNP formulations showed increased particle size and decreased encapsulation efficiencies. An increasing molar ratio of poly-(ethylene) glycol (PEG)-lipid significantly decreased size but also intracellular protein expression of mRNA. We identified four formulations possessing higher intracellular protein expression ability in vitro even after aerosolization which were then assessed in in vivo studies. It was found that luciferase protein was predominately expressed in the mouse lung for the four lead formulations before and after nebulization. This study demonstrated that LNPs hold promise to be applied for aerosolization-mediated pulmonary mRNA delivery.

Controlled loading of albumin-drug conjugates ex vivo for enhanced drug delivery and antitumor efficacy.

To harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Here, model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drug would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native secondary structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that better recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to better maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy.

Just how prevalent are peptide therapeutic products? A critical review.

How prevalent are peptide therapeutic products? How innovative are the formulations used to deliver peptides? This review provides a critical analysis of therapeutic peptide products and the formulations approved by the United States Food and Drug administration (FDA), the European Medicines Agency (EMA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). This review also provides an in-depth analysis of dosage forms and administration routes for delivering peptide therapeutics, including injectables, oral dosage forms, and other routes of administration. We discuss the function of excipients in parenteral formulations in detail, since most peptide therapeutics are parenterally administered. We provide case studies of alternate delivery routes and dosage forms. Based on our analysis, therapeutic peptides administered as injectables remain the most commonly used dosage forms, particularly in the form of subcutaneous, intravenous, or intramuscular injections. In addition, therapeutic peptides are formulated to achieve prolonged release, often through the use of polymer carriers. The limited number of oral therapeutic peptide products and their poor absorption and subsequent low bioavailability indicate a need for new technologies to broaden the formulation design space. Therapeutic peptide products may also be delivered through other administration routes, including intranasal, implant, and sublingual routes. Therefore, an in-depth understanding of how therapeutic peptides are now formulated and administered is essential to improve peptide delivery, improve patient compliance, and reduce the healthcare burden for these crucial therapeutic agents.

Electrostatic driven transport enhances penetration of positively charged peptide surfaces through tumor extracellular matrix.

Drug carriers achieve poor and heterogeneous distribution within solid tumors due to limited transport through the tumor extracellular matrix (ECM). The tumor ECM forms a net negatively charged network that interacts with and hinders the transport of molecules in part due to electrostatic interactions. Traditionally, the surfaces of drug delivery systems are passivated to minimize these interactions, but the mechanism of how charge interactions impact transport and penetration within the tumor microenvironment (TME) is not well understood. Here, we used T7 bacteriophage as a model biological nanoparticle to display peptides of different charges on its surface and elucidate how charge-based binding drives transport, uptake, and retention within tumor tissue. In contrast to current studies with neutrally charged surfaces, we discovered that a positively charged peptide displayed on T7 enhanced its penetration through a tumor-like ECM when compared to neutrally and negatively charged peptides. The positively charged peptide displayed on T7 facilitated weak and reversible binding with the TME to achieve Donnan partitioning and deep penetration into ex vivo tumor tissue. Additionally, the positively charged peptide-presenting T7 has a high number of intra-tissue binding sites in the TME (~4 µM) that enables almost 100% retention in the tumor tissue for up to 24 h. These results, coupled with transport studies of systematically mutated T7, show that electrostatic interactions can be responsible for uptake and retention of the positively charged peptide-presenting T7 within the net negatively charged TME. STATEMENT OF SIGNIFICANCE: The TME selectively hinders the transport of drugs and drug delivery systems due to their size, shape, and intermolecular interactions. Typically, the focus in drug delivery has been to develop delivery systems smaller than the pore size of the tumor ECM and/or develop inert surface coatings that have negligible interactions with the tumor ECM for diffusive transport. While there is an association of the surface charge of carriers with their transport through the tumor ECM, the mechanism of charge-driven transport is poorly understood. In this work, we elucidate the mechanism and find that interestingly, particles with a weakly positive surface charge interact with the net negatively charged tumor ECM to significantly improve their uptake, penetration, and retention in tumor tissue.

The Stabilizing Excipients in Dry State Therapeutic Phage Formulations.

Phage therapy has gained prominence due to the increasing pathogenicity of "super bugs" and the rise of their multidrug resistance to conventional antibiotics. Dry state formulation of therapeutic phage is attractive to improve their "druggability" by increasing their shelf life, improving their ease of handling, and ultimately retaining their long-term potency. The use and selection of excipients are critical to stabilize phage in solid formulations and protect their viability from stresses encountered during the solidification process and long-term storage prior to use. Here, this review focuses on the current classes of excipients used to manufacture dry state phage formulations and their ability to stabilize and protect phage throughout the process, as discussed in the literature. We provide perspective of outstanding challenges involved in the formulation of dry state phage. We suggest strategies to improve excipient identification and selection, optimize the potential excipient combinations to improve phage viability during formulation, and evaluate new methodologies that can provide greater insight into phage-excipient interactions to improve design criteria to improve formulation of dry state phage therapeutics. Addressing these challenges opens up new opportunities to re-design and re-imagine phage formulations for improved efficacy as a pharmaceutical product.

Peptides as surface coatings of nanoparticles that penetrate human cystic fibrosis sputum and uniformly distribute in vivo following pulmonary delivery.

Therapeutic delivery of drug and gene delivery systems have to traverse multiple biological barriers to achieve efficacy. Mucosal administration, such as pulmonary delivery in cystic fibrosis (CF) disease, remains a significant challenge due to concentrated viscoelastic mucus, which prevents drugs and particles from penetrating the mucus barrier. To address this problem, we used combinatorial peptide-presenting phage libraries and next-generation sequencing (NGS) to identify hydrophilic, net-neutral charged peptide coatings that enable penetration through human CF mucus ex vivo with ~600-fold better penetration than control, improve uptake into lung epithelial cells compared to uncoated or PEGylated-nanoparticles, and exhibit enhanced uniform distribution and retention in the mouse lung airways. These peptide coatings address multiple delivery barriers and effectively serve as excellent alternatives to standard PEG surface chemistries to achieve mucus penetration and address some of the challenges encountered using these chemistries. This biomolecule-based strategy can address multiple delivery barriers and hold promise to advance efficacy of therapeutics for diseases like CF.

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment.

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts as a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) affect their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and quantify their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and spatial ordering impact diffusive transport. The P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited ∼40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to functionalized control particles. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.

Intracellular nanoparticle delivery by oncogenic KRAS-mediated macropinocytosis.

BACKGROUND: The RAS family of oncogenes (KRAS, HRAS, NRAS) are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression. As a result, drug delivery targeting RAS-driven tumors has been a long-standing challenge in cancer therapy. Mutant RAS activates cancer cells to actively take up nutrients, including glucose, lipids, and albumin, via macropinocytosis to fulfill their energetic requirements to survive and proliferate. PURPOSE: We exploit macropinocytosis pathway to deliver nanoparticles (NPs) in cancer cells harboring activating KRAS mutations. METHODS: NPs were synthesized by the desolvation method. The physicochemical properties and stability of NPs were characterized by dynamic light scattering and transmission electron microscopy. Uptake of fluorescently labelled NPs in wild-type and mutant KRAS cells were quantitively determined by flow cytometry and qualitatively by fluorescent microscopy. NP uptake by KRAS-driven macropinocytosis was confirmed by pharmacological inhibition and genetic knockdown. RESULTS: We have synthesized stable albumin NPs that demonstrate significantly greater uptake in cancer cells with activating mutations of KRA S than monomeric albumin (ie, dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these NPs exhibit significantly increased uptake in mutant KRAS cancer cells than wild-type KRAS cells by macropinocytosis. CONCLUSIONS: The uptake of albumin nanoparticles is driven by KRAS. This NP-based strategy targeting RAS-driven macropinocytosis is a facile approach toward improved delivery into KRAS-driven cancers.

Quantitative PCR of T7 Bacteriophage from Biopanning.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., "biopanning"). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2-4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g., immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

Mucus-penetrating phage-displayed peptides for improved transport across a mucus-like model.

The objective of this work is to use phage display libraries as a screening tool to identify peptides that facilitate transport across the mucus barrier. Mucus is a complex selective barrier to particles and molecules, limiting penetration to the epithelial surface of mucosal tissues. In mucus-associated diseases such as cystic fibrosis (CF), mucus has increased viscoelasticity and a higher concentration of covalent and non-covalent physical entanglements compared to healthy tissues, which greatly hinders permeability and transport of drugs and particles across the mucosae for therapeutic delivery. Treatment of CF lung diseases and associated infections must overcome this abnormal mucosal barrier. Critical bottlenecks hindering effective drug penetration remain and while recent studies have shown hydrophilic, net-neutral charge polymers can improve the transport of nanoparticles and minimize interactions with mucus, there is a dearth of alternative carriers available. We hypothesized that the screening of a phage peptide library against a CF mucus model would lead to the identification of phage-displayed peptide sequences able to improve transport in mucus. These combinatorial libraries possess a large diversity of peptide-based formulations (108-109) to achieve unprecedented screening for potential mucus-penetrating peptides. Here, phage clones displaying discovered peptides were shown to have up to 2.6-fold enhanced diffusivity in the CF mucus model. In addition, we demonstrate reduced binding affinities to mucin compared to wild-type control. These findings suggest that phage display libraries can be used as a strategy to improve transmucosal delivery.

Peptides as drug delivery vehicles across biological barriers.

Peptides are small biological molecules that are attractive in drug delivery and materials engineering for applications including therapeutics, molecular building blocks and cell-targeting ligands. Peptides are small but can possess complexity and functionality as larger proteins. Due to their intrinsic properties, peptides are able to overcome the physiological and transport barriers presented by diseases. In this review, we discuss the progress of identifying and using peptides to shuttle across biological barriers and facilitate transport of drugs and drug delivery systems for improved therapy. Here, the focus of this review is on rationally designed, phage display peptides, and even endogenous peptides as carriers to penetrate biological barriers, specifically the blood-brain barrier(BBB), the gastrointestinal tract (GI), and the solid tumor microenvironment (T). We will discuss recent advances of peptides as drug carriers in these biological environments. From these findings, challenges and potential opportunities to iterate and improve peptide-based approaches will be discussed to translate their promise towards the clinic to deliver drugs for therapeutic efficacy.

Manufacturing and ambient stability of shelf freeze dried bacteriophage powder formulations.

The severity of multidrug resistance to antibiotics has urged development of alternative treatment approaches, including bacteriophage therapy. Given the complexity of the bacteriophage structure, formulation and stability are primary concerns. Our present work optimized process and formulations of phage powder manufacturing and investigated the stability of lyophilized bacteriophage powders under ambient storage. The model phage M13 was formulated with trehalose, mannitol, sucrose and PEG6000 and lyophilized in different conditions. Bacteriophage viability was examined by titering and was considered as the assessment of phage stability. Less titer loss of trehalose and sucrose formulations were observed compared to mannitol and PEG groups both immediately after lyophilization and upon long term storage. When evaluating lyophilization conditions, an additional 1 log titer was preserved by reduction of product drying stress. Trehalose was stabilized in the amorphous state whereas mannitol stayed in crystalline state in lyophilized powders. Increased moisture content was demonstrated to have a positive impact on viability of phage after lyophilization and upon storage. Overall, 2% trehalose or sucrose (w/v) can sufficiently stabilize phage during lyophilization process and storage in ambient conditions. There is a positive correlation between residual water and stability of phage. These collective findings highlight the potential of long-term, ambient storage of bacteriophage towards their successful use in diverse healthcare settings.

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*101-2.75*108genome copies(gc)/µL and 2.66*101-2.66*108 genome copies(gc)/µL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (Es) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*107gc/µL and 2.66*101-2.66*107gc/µL of M13 and T7 phage DNA, with an amplification efficiency Es of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS).

Physicochemical properties of mucus and their impact on transmucosal drug delivery.

Mucus is a selective barrier to particles and molecules, preventing penetration to the epithelial surface of mucosal tissues. Significant advances in transmucosal drug delivery have recently been made and have emphasized that an understanding of the basic structure, viscoelastic properties, and interactions of mucus is of great value in the design of efficient drug delivery systems. Mucins, the primary non-aqueous component of mucus, are polymers carrying a complex and heterogeneous structure with domains that undergo a variety of molecular interactions, such as hydrophilic/hydrophobic, hydrogen bonds and electrostatic interactions. These properties are directly relevant to the numerous mucin-associated diseases, as well as delivering drugs across the mucus barrier. Therefore, in this review we discuss regional differences in mucus composition, mucus physicochemical properties, such as pore size, viscoelasticity, pH, and ionic strength. These factors are also discussed with respect to changes in mucus properties as a function of disease state. Collectively, the review seeks to provide a state of the art roadmap for researchers who must contend with this critical barrier to drug delivery.

Deep, noninvasive imaging and surgical guidance of submillimeter tumors using targeted M13-stabilized single-walled carbon nanotubes.

Highly sensitive detection of small, deep tumors for early diagnosis and surgical interventions remains a challenge for conventional imaging modalities. Second-window near-infrared light (NIR2, 950-1,400 nm) is promising for in vivo fluorescence imaging due to deep tissue penetration and low tissue autofluorescence. With their intrinsic fluorescence in the NIR2 regime and lack of photobleaching, single-walled carbon nanotubes (SWNTs) are potentially attractive contrast agents to detect tumors. Here, targeted M13 virus-stabilized SWNTs are used to visualize deep, disseminated tumors in vivo. This targeted nanoprobe, which uses M13 to stably display both tumor-targeting peptides and an SWNT imaging probe, demonstrates excellent tumor-to-background uptake and exhibits higher signal-to-noise performance compared with visible and near-infrared (NIR1) dyes for delineating tumor nodules. Detection and excision of tumors by a gynecological surgeon improved with SWNT image guidance and led to the identification of submillimeter tumors. Collectively, these findings demonstrate the promise of targeted SWNT nanoprobes for noninvasive disease monitoring and guided surgery.