Purpose Long-acting formulations of the potent antiretroviral prodrug tenofovir alafenamide (TAF) hold potential as biomedical HIV prevention modalities. Here, we present a rigorous comparison of three animal models, C57BL/6 J mice, beagle dogs, and merino sheep for evaluating TAF implant pharmacokinetics (PKs). Methods Implants delivering TAF over a wide range of controlled release rates were tested in vitro and in mice and dogs. Our existing PK model, supported by an intravenous (IV) dosing dog study, was adapted to analyze mechanistic aspects underlying implant TAF delivery. Results TAF in vitro release in the 0.13 to 9.8 mg d −1 range with zero order kinetics were attained. Implants with equivalent fabrication parameters released TAF in mice and sheep at rates that were not statistically different, but were 3 times higher in dogs. When two implants were placed in the same subcutaneous pocket, a two-week creep to C max was observed in dogs for systemic drug and metabolite concentrations, but not in mice. Co-modeling IV and TAF implant PK data in dogs led to an apparent TAF bioavailability of 9.6 in the single implant groups (compared to the IV group), but only 1.5 when two implants were placed in the same subcutaneous pocket. Conclusions Based on the current results, we recommend using mice and sheep, with macaques as a complementary species, for preclinical TAF implant evaluation with the caveat that our observations may be specific to the implant technology used here. Our report provides fundamental, translatable insights into multispecies TAF delivery via long-acting implants.

Global efforts aimed at preventing human immunodeficiency virus type one (HIV-1) infection in vulnerable populations appear to be stalling, limiting our ability to control the epidemic. Long-acting, controlled drug administration from subdermal implants holds significant potential by reducing the compliance burden associated with frequent dosing. We, and others, are exploring the development of complementary subdermal implant technologies delivering the potent prodrug, tenofovir alafenamide (TAF). The current report addresses knowledge gaps in the preclinical pharmacology of long-acting, subdermal TAF delivery using several mouse models. Systemic drug disposition during TAF implant dosing was explained by a multi-compartment pharmacokinetic (PK) model. Imaging mass spectrometry was employed to characterize the spatial distribution of TAF and its principal five metabolites in local tissues surrounding the implant. Humanized mouse studies determined the effective TAF dose for preventing vaginal and rectal HIV-1 acquisition. Our results represent an important step in the development of a safe and effective TAF implant for HIV-1 prevention.

Mixed-valent iron-layered double hydroxides (LDHs), also known as "green rust" (GR), are increasingly becoming recognized as important chemically reduced minerals. GR is believed to have contributed to the accumulation of biomolecules in primordial oceans and to have played a role in the geochemical cycles of early Earth and Mars. The high oxygen sensitivity of these LDHs makes them challenging to prepare and study. Consequently, a synthesis is described herein that affords pure GR compounds over a range of Fe(III) mole fractions and with a variety of intercalated anions. Reliable synthesis of pure GR required two key considerations: (1) ensure accurate and controlled pH titrations and (2) rigorously exclude oxygen before, during, and after the target Fe(II)/Fe(III) ratio is achieved. A detailed protocol for the strictly anoxic synthesis, real-time reaction monitoring, product characterization, and transport of a variety of GR samples is described. Protection of the air-sensitive samples from traces of oxygen for postsynthesis measurements or experiments can be challenging, leading to the potential formation of oxidation product impurities. Analysis of GR specimens by electron microscopy with the described methods enables rapid quality control to confirm sample integrity, including elemental microanalysis. The experimental details in the preparation of pure GR samples provide an important foundation to the geochemical community studying this chemically versatile group of compounds.

Background:The equilibrative nucleoside transporter 1 (ENT1), a protein encoded by the SLC29A1 gene, is able to carry nucleosides across cell membranes.The high affinity for adenosine makes it relevant to anti-retroviral therapy (ART), where the drugs are adenosine analogs.The ENT1 protein might transport adenosine drug analogs such as tenofovir (TFV) and its prodrugs into and out of cells, removing them from their site of action.Methods: A commercial polyclonal antibody to the ENT1 protein was applied to a vaginal cultured cell line and on vaginal epithelial cells (VEC) donated by female volunteers.Antibody labeling was tested by western blotting, by light and electron microscopy, and by quantitative analysis of anti-ENT1 label on V19 multilayer cultures.Antibody specificity was tested by affinity adsorption.Results: The ENT1 protein was present on VEC from human donors and on V19 cultured cells.The ENT1 antibody revealed protein bands at 50 kDa and 30 kDa on a western blot of V19 proteins.Light microscopy revealed only limited information on the sub-cellular location of the ENT1 protein, but immuno-electron microscopy showed the ENT1 protein predominantly located on the plasma membrane and lysosome-like structures.Label was also present over the endoplasmic reticulum (ER), Golgi complex, and cytoplasmic vesicles. Conclusions:The results suggest a synthesis and degradation pathway for the ENT1 protein.However, the presence of ENT1 in junctional complexes and mitochondria suggest a more complex role for the protein.The ENT1 protein may potentially transport adenosine drug analogs such as tenofovir (TFV) and its prodrugs into and out of cells.The presence of ENT1 on VEC, with a rapid turnover, may have wideranging implications on anti-retroviral treatment (ART) therapies by removing them from their site of action.However, the bidirectional nature of ENT1 could mean that drugs such as TFV are also removed from cells.Transporter proteins such as ENT1 could shuttle the TFV through the vaginal epithelium to immune cells located in lower cell layers.Future studies will focus on determining if the ENT1 protein is active on vaginal cell plasma membranes, and if it has a role in the transport of nucleoside analogs.

Understanding the structure and behavior of chemical gardens is of interest for materials science, for understanding organic-mineral interactions, and for simulating geological mineral structures in hydrothermal systems on Earth and other worlds. Herein, we explored the effects of amino acids on inorganic chemical garden precipitate systems of iron chloride and sodium silicate to determine if/how the addition of organics can affect self-assembling morphologies or crystal growth. Amino acids affect chemical garden growth and morphology at the macro-scale and at the nanoscale. In this reaction system, the concentration of amino acid had a greater impact than the amino acid side chain, and increasing concentrations of organics caused structures to have smoother exteriors as amino acids accumulated on the outside surface. These results provide an example of how organic compounds can become incorporated into and influence the growth of inorganic self-organizing precipitates in far-from-equilibrium systems. Additionally, sample handing methods were developed to successfully image these delicate structures.

New HIV-1 infection rates far outpace the targets set by global health organizations, despite important progress in curbing the progression of the epidemic. Long-acting (LA) formulations delivering antiretroviral (ARV) agents for HIV-1 pre-exposure prophylaxis (PrEP) hold significant promise, potentially facilitating adherence due to reduced dosing frequency compared to oral regimens. We have developed a subdermal implant delivering the potent ARV drug tenofovir alafenamide that could provide protection from HIV-1 infection for 6 months, or longer. Implants from the same lot were investigated in mice and sheep for local safety and pharmacokinetics (PKs). Ours is the first report using these animal models to evaluate subdermal implants for HIV-1 PrEP. The devices appeared safe, and the plasma PKs as well as the drug and metabolite concentrations in dermal tissue adjacent to the implants were studied and contrasted in two models spanning the extremes of the body weight spectrum. Drug and drug metabolite concentrations in dermal tissue are key in assessing local exposure and any toxicity related to the active agent. Based on our analysis, both animal models were shown to hold significant promise in LA product development.

Transporters are specialized integral membrane proteins, which mediate the passage of virtually all molecules through cell membranes. They are expressed in a broad range of human and animal tissues and play important roles in both normal and disease states. For these reasons, they are evaluated when developing and testing drugs. Two major families of drug transporters, the adenosine 5'-triphosphate-binding cassette and solute carrier transporters (SLC), have critical roles in the absorption, distribution, metabolism, and elimination of drugs. The SLC family contains known nucleoside transporters and therefore are important when nucleoside analogs are used as drugs to prevent or treat viral infections. In this study, we wanted to determine if it was possible to locate one member of the SLC family, the human concentrative nucleoside transporter 3 (CNT3) in human vaginal epithelial cells. The CNT3 protein has important roles in drug delivery, subsequent drug tissue distribution, and, hence, efficacy. Vaginal epithelial cells, taken from two human volunteers (one Caucasian and one African American), were labeled for light and electron microscopy, with a commercial antibody to a cytoplasmic domain of CNT3, the protein product of the SLC28A3 gene. Fluorescent secondary antibodies or protein A-gold were used to detect antibody binding. By electron microscopy, gold particle binding was quantified to determine labeling specificity. By light microscopy, positive labeling with anti-CNT3 antibodies was detected on human vaginal epithelial cells, but specificity to any intracellular structure was not easily determined, most likely a result of specimen preparation. Electron microscopy revealed that the CNT3 transporter protein was present predominantly on microvilli located on one side of some human vaginal epithelial cells. Quantification confirmed specific anti-CNT3 labeling over human vaginal epithelial cell microvilli. The CNT3 protein, present in the microvilli of human vaginal epithelial cells, may have a role in redistributing nucleoside homologues delivered to the vaginal tract. Transporter proteins such as CNT3 could shuttle nucleosides and their analogs through the vaginal epithelium to immune cells located in lower cell layers. Outer layers of cells, which are eventually shed from the epithelium, may remove accumulated nucleoside drug analogs from the vaginal tract.

Optimal pre-analytical handling is essential for valid measurements of plasma concentration and size distribution of extracellular vesicles (EVs). We investigated the impact of plasma preparation, various anticoagulants (Citrate, EDTA, CTAD, Heparin), and fasting status on concentration and size distribution of EVs measured by Nanoparticle Tracking Analysis (NTA). Blood was drawn from 10 healthy volunteers to investigate the impact of plasma preparation and anticoagulants, and from 40 individuals from a population-based study to investigate the impact of postprandial lipidemia. Plasma concentration of EVs was measured by NTA after isolation by high-speed centrifugation, and size distribution of EVs was determined using NTA and scanning electron microscopy (SEM). Plasma concentrations and size distributions of EVs were essentially similar for the various anticoagulants. Transmission electron microscopy (TEM) confirmed the presence of EVs. TEM and SEM-analyses showed that the EVs retained spherical morphology after high-speed centrifugation. Plasma EVs were not changed in postprandial lipidemia, but the mean sizes of VLDL particles were increased and interfered with EV measurements (explained 66% of the variation in EVs-concentration in the postprandial phase). Optimization of procedures for separating VLDL particles and EVs is therefore needed before NTA-assessment of EVs can be used as biomarkers of disease.

Nanoparticle-based therapeutics are being used to treat patients with solid tumors. Whereas nanoparticles have been shown to preferentially accumulate in solid tumors of animal models, there is little evidence to prove that intact nanoparticles localize to solid tumors of humans when systemically administered. Here, tumor and adjacent, nonneoplastic tissue biopsies are obtained through endoscopic capture from patients with gastric, gastroesophageal, or esophageal cancer who are administered the nanoparticle CRLX101. Both the pre- and postdosing tissue samples adjacent to tumors show no definitive evidence of either the nanoparticle or its drug payload (camptothecin, CPT) contained within the nanoparticle. Similar results are obtained from the predosing tumor samples. However, in nine of nine patients that were evaluated, CPT is detected in the tumor tissue collected 24-48 h after CRLX101 administration. For five of these patients, evidence of the intact deposition of CRLX101 nanoparticles in the tumor tissue is obtained. Indications of CPT pharmacodynamics from tumor biomarkers such as carbonic anhydrase IX and topoisomerase I by immunohistochemistry show clear evidence of biological activity from the delivered CPT in the posttreatment tumors.

Background: Nanoparticle-based therapeutics are thought to rely on the enhanced permeability and retention effect to preferentially localize in solid tumors and not healthy tissue. These phenomena are rationalized primarily from animal models of the human disease. There is a need to obtain analogous information from humans in order to better understand how nanoparticle-based therapeutics perform in humans. CRLX101 is an investigational nanoparticle drug conjugate (NDC) consisting of a cyclodextrin-containing polymer (CDP) conjugate of the payload camptothecin (CPT). The individual polymer strands self-assemble into nanoparticles (ca. five strands) of approximately 10 to 40 nm diameter and 10 wt% CPT by multiple, interstrand, inclusion complex formation between the cyclodextrin and the CPT molecules. CRLX101 is currently being investigated in phase II trials in patients with renal, ovarian and rectal cancer. Methods: A phase I clinical trial was performed with CRLX101 at the City of Hope, in patients with advanced or metastatic stomach, gastroesophageal or esophageal cancer. This study was sponsored by City of Hope Medical Center, and funding and CRLX101 was provided by Cerulean Pharma Inc. (ClinicalTrials.gov identifier: NCT01612546). The goal of this study was to test the hypothesis that intact CRLX101 nanoparticles deposit in human tumors and not in normal adjacent tissue after intravenous administration. Tumor and adjacent healthy tissue biopsies were obtained through endoscopic capture from patients who received CRLX101, and analyzed via a number of methodologies. Results: Both the pre- and post-dosing, healthy tissue samples adjacent to tumors show no evidence of either the NDC or the payload (CPT) contained within the NDC. Similar results are obtained from the pre-dosing tumor samples. However, in 8 of 9 patients that were evaluated, CPT is detected in the tumor tissue by fluorescent microscopy examination of fixed sections. For 3 of these patients, proof of intact CRLX101 NDC is obtained from tissue sections by colocalized, fluorescence from the CPT and a secondary antibody used to stain a PEG specific antibody (binds to the PEG in CRLX101). Following fluorescence imaging, remaining tissues from 3 patients were homogenized and measured for free and CDP conjugated CPT using HPLC. CPT was present in the post-treatment tumor tissue of all 3 patient samples with an average of 96.2±13.1% in the conjugated form, indicating that CRLX101 NDCs are localized in these samples. Conclusions: Tumor and adjacent healthy tissue biopsies obtained from cancer patients who have received CRLX101show that these NDCs do localize in human tumors and not in adjacent tissues. Citation Format: Andrew Clark, Devin T. Wiley, Jonathan E. Zuckerman, Paul Webster, Joseph Chao, James Lin, Yun Yen, Mark E. Davis, Scott Eliasof. CRLX101, an investigational nanoparticle-drug conjugate, localizes in human tumors and not in adjacent healthy tissue after intravenous dosing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B33.

In July 2015 Professor K.T. Tokuyasu passed away in San Diego giving us the opportunity to reflect on the contribution this electron microscopist made to the field of immunocytochemistry. His work provided a sensitive, minimally invasive approach to producing thin sections of biological material for labeling with antibodies. His approach has been applied to a wide range of biological applications and provided important information on cellular processes.

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheet-like structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand-containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity.

Unraveling the complex ecology of the vaginal biofilm microbiome relies on a number of complementary techniques. Here, we describe the experimental approaches for studying vaginal microbial biofilm samples with a focus on specimen preparation for subsequent analysis. The techniques include fluorescence microscopy, fluorescence in situ hybridization, and scanning and transmission electron microscopy. Isolation of microbial DNA and RNA from these samples is covered along with a brief discussion of chemical analysis methods.

Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.