Penetration Of Small Molecules Research Articles

Controlling Anti-Penetration Performance by Post-Grafting of Fluorinated Alkyl Chains onto Polystyrene-block-poly(vinyl methyl siloxane).

Polydimethylsiloxane (PDMS) has been widely used as a surface coating material, which has been reported to possess dynamic omniphobicity to a wide range of both polar and nonpolar solvents due to its high segmental flexibility and mobility. However, such high flexibility and mobility also enable penetration of small molecules into PDMS coatings, which alter the chemical and physical properties of the coating layers. To improve the anti-penetration properties of PDMS, a series of fluorinated alkyl segments are grafted to a diblock copolymer of polystyrene-block-poly(vinyl methyl siloxane) (PS-b-PVMS) using thiol-ene click reactions. This article reports the chemical characterization of these model fluorosilicone block copolymers and uses fluorescence measurements to investigate the dye penetration characteristics of polymer thin films. The introduction of longer fluorinated alkyl chains can gradually increase the anti-penetration properties as the time to reach the maximum fluorescence intensity (tpeak) gradually increases from 11 s of PS-b-PVMS to more than 1000 s of PS-b-P(n-C6F13-VMS). The improvement of anti-penetration properties is attributed to stronger inter-/intrachain interactions, phase segregation of ordered fluorinated side chains, and enhanced hydrophobicity caused by the grafting of fluorinated alkyl chains.

Abstract 4774: Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26

Abstract Current challenges for pancreatic ductal adenocarcinoma (PDAC) treatment include overcoming the disease’s fibrous tumor microenvironment with limited cytotoxicity- in which tissue stiffening is believed to occur even pre-disease onset. Acinar to ductal metaplasia (ADM), the transdifferentiation of acinar cells into duct-like cells, is a predisposing event to PDAC development that is also linked to Hippo signaling, a major pathway involved in fibrosis and inflammation. Hippo activation is initiated by the yes-associated protein (YAP) in conjunction with the PDAZ binding motif (TAZ) and TEA domain (TEAD) transcription factor. Thus, the synthesis of the novel covalent TEAD autopalmitoylation inhibitor, CV-4-26, presented an opportunity to therapeutically target ADM. CV-4-26 binds to the TEAD lipid binding pocket, which, in turns, hinders TEAD palmitoylation, thereby leading to the inhibition of the Hippo pathway. Utilizing our 3D organoid p48Cre/+LSL-KRASG12D/+ (KC) mouse model, we demonstrated pronounced phenotypic and molecular inhibition of ADM by CV-4-26, resulting in a conserved acinar phenotype, as well as downregulation of downstream markers of the YAP/TAZ complex. The compound did not reverse ADM to the acinar state once ducts were visibly formed. Yet, CV-4-26 demonstrated a lack of cytotoxicity (up to 25 µM) as determined by Calcein AM staining. Biomechanical measurements of extracellular matrix stiffness acquired through calculations of fluorescent nanoparticle displacement further displayed decreased moduli as a response to inhibition treatment. Moreover, a 56-fold downregulation of collagen (COL1A1, COL1A2) mRNA, markers for PDAC development, was observed. However, less reduction in the collagens and extracellular matrix stiffness was observed in the ADM reversal treatments, corroborating our morphology and gene expression results. Our data suggest that combining CV-4-26 by ADM inhibition with standard of care therapies for PDAC may enhance the penetration of small molecules through the tumor’s extracellular matrix. Citation Format: Corey M. Perkins, Chen Zhou, Martha Campbell-Thompson, Yating Mao, Kalina R. Atanasova, Jinmai Jiang, Ranjala Ratnayake, Hendrik Luesch, Jamel Ali, Chenglong Li, Thomas D. Schmittgen. Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4774.

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules.

In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood-brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.

Non-Invasive Transdermal Delivery of Antisense Oligonucleotides with Biocompatible Ionic Liquids.

Nucleic acid drugs, including antisense oligonucleotides (ASOs), have received considerable attention as novel therapeutics for the treatment of intractable diseases. Despite their potential benefits, ASOs are currently administered via injection, which can negatively impact patient quality of life because of the prevalence of severe injection site reactions. Non-invasive transdermal administration of ASOs is desirable but highly challenging owing to the strong barrier imposed by the stratum corneum, which only permits the penetration of small molecules under 500 Da. For ASOs to exert their antisense effect, they must traverse the negatively charged cell membrane and reach the cytoplasm. In this study, we used the solid-in-oil (S/O) dispersion technology to facilitate the skin permeation of ASOs by coating the drug with a hydrophobic surfactant molecule, specifically lipid-based ionic liquid (IL) surfactants with high biocompatibility and transdermal penetration-enhancing properties. To induce the antisense effect, it was important to achieve simultaneous transdermal delivery and intracellular entrapment of ASOs. In vitro investigations indicated that the newly prepared IL-S/O enhanced the transdermal penetration and intracellular delivery of ASOs, thus inhibiting mRNA translation of the target TGF-β. In addition, in vivo investigations of tumor-bearing mice suggested that the anti-tumor effect of the IL-S/O was similar to that of injection. This study demonstrates the potential of non-invasive transdermal delivery carriers based on biocompatible ILs, which can be applied to a variety of nucleic acid drugs.

Optimizing transcardial perfusion of small molecules and biologics for brain penetration and biodistribution studies in rodents.

Efficiently removing blood from the brain vasculature is critical to evaluate accurately the brain penetration and biodistribution of drug candidates, especially for biologics as their blood concentrations are substantially higher than the brain concentrations. Transcardial perfusion has been used widely to remove residual blood in the brain; however, the perfusion conditions (such as the perfusion rate and time) reported in the literature are quite varied, and the performance of these methods on blood removal has not been investigated thoroughly. In this study, the effectiveness of the perfusion conditions was assessed by measuring brain hemoglobin levels. Sodium nitrite (NaNO2 ) as an additive in the perfusate was evaluated at different concentrations. Blood removal was significantly improved with 2% NaNO2 over a 20 min perfusion in mouse without disrupting the integrity of the blood-brain barrier (BBB). In mice, the optimized perfusion method significantly lowered the measured brain-to-plasma ratio (Kp,brain ) for monoclonal antibodies due to the removal of blood contamination and small molecules with a moderate-to-high BBB permeability and with a high brain-unbound-fraction (fu,brain ) presumably due to flux out of the brain during perfusion. Perfusion with or without NaNO2 clearly removed the residual blood in rat brain but with no difference observed in Kp,brain between the perfusion groups with or without 2% NaNO2 . In conclusion, a perfusion method was successfully developed to evaluate the brain penetration of small molecules and biologics in rodents for the first time. The transcardial perfusion with 2% NaNO2 effectively removed the residual blood in the brain and significantly improved the assessment of brain penetration of biologics. For small molecules, however, transcardial perfusion may not be performed, as small molecule compounds could be washed away from the brain by the perfusion procedure.

Silencing PCBP2 normalizes desmoplastic stroma and improves the antitumor activity of chemotherapy in pancreatic cancer.

Rationale: Dense desmoplastic stroma is a fundamental characteristic of pancreatic ductal adenocarcinoma (PDAC) and comprises up to 80% of the tumor mass. Type I collagen is the major component of the extracellular matrix (ECM), which acts as a barrier to impede the delivery of drugs into the tumor microenvironment. While the strategy to deplete PDAC stroma has failed in clinical trials, normalization of the stroma to allow chemotherapy to kill the tumor cells in the “nest” could be a promising strategy for PDAC therapy. We hypothesize that silencing the poly(rC)-binding protein 2 (αCP2, encoded by the PCBP2 gene) leads to the destabilization and normalization of type I collagen in the PDAC stroma.Methods: We develop a micro-flow mixing method to fabricate a peptide-based core-stabilized PCBP2 siRNA nanocomplex to reverse the accumulation of type I collagen in PDAC tumor stroma. Various in vitro studies were performed to evaluate the silencing activity, cellular uptake, serum stability, and tumor penetration of the PCBP2 siRNA nanocomplex. We also investigated the penetration of small molecules in stroma-rich pancreatic cancer spheroids after the treatment with the PCBP2 siRNA nanocomplex. The anti-tumor activity of the PCBP2 siRNA nanocomplex and its combination with gemcitabine was evaluated in an orthotopic stroma-rich pancreatic cancer mouse model.Results: Silencing the PCBP2 gene using siRNA reverses the accumulation of type I collagen in human pancreatic stellate cells (PSCs) and mouse NIH 3T3 fibroblast cells. The siRNA nanocomplex significantly reduces ECM production and enhances drug penetration through desmoplastic tumor stroma. The combination of gemcitabine with the PCBP2 siRNA nanocomplex markedly suppresses the tumor progression in a desmoplastic PDAC orthotopic mouse model.Conclusion: This approach provides a new therapeutic avenue to improve the antitumor efficacy of PDAC therapies by normalizing tumor stroma using the PCBP2 siRNA nanocomplex.

In vivo quantitative molecular absorption of glycerol in human skin using coherent anti-Stokes Raman scattering (CARS) and two-photon auto-fluorescence

The penetration of small molecules through the human skin is a major issue for both safety and efficacy issues in cosmetics and pharmaceutic domains. To date, the quantification of active molecular compounds in human skin following a topical application uses ex vivo skin samples mounted on Franz cell diffusion set-up together with appropriate analytical methods. Coherent anti-Stokes Raman scattering (CARS) has also been used to perform active molecule quantification on ex vivo skin samples, but no quantification has been described in human skin in vivo. Here we introduce and validate a framework for imaging and quantifying the active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework was exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time was demonstrated, showing that, contrary to water, the xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time. More generally, the proposed imaging framework provides an enabling platform for establishing functional activity of topically applied products in vivo.

Preferential Equilibrium Partitioning of Positively Charged Tryptophan into Phosphatidylcholine Bilayer Membranes.

The interactions between small molecules and lipid bilayers play a critical role in the function of cellular membranes. Understanding how a small molecule interacts with the lipid bilayer differently based on its charge reveals primordial mechanisms of transport across membranes and assists in the design of drug molecules that can penetrate cells. We have previously reported that tryptophan permeated through a phosphatidylcholine lipid bilayer membrane at a faster rate when it was positively charged (Trp+) than when negatively charged (Trp−), which corresponded to a lower potential of mean force (PMF) barrier determined through simulations. In this report, we demonstrate that Trp+ partitions into the lipid bilayer membrane to a greater degree than Trp– by interacting with the ester linkage of a phosphatidylcholine lipid, where it is stabilized by the electron withdrawing glycerol functional group. These results are in agreement with tryptophan’s known role as an anchor for transmembrane proteins, though the tendency for binding of a positively charged tryptophan is surprising. We discuss the implications of our results on the mechanisms of unassisted permeation and penetration of small molecules within and across lipid bilayer membranes based on molecular charge, shape, and molecular interactions within the bilayer structure.

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules.

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules in vivo and in vitro, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an ex vivo method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after in vitro incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides.

The mechanism of plasma-assisted penetration of NO2− in model tissues

Cold atmospheric plasmas are reportedly capable of enhancing the percutaneous absorption of drugs, which is a development direction of plasma medicine. This motivated us to study how the enhancement effect was realized. In this letter, gelatin gel films were used as surrogates of human tissues, NaNO2 was used as a representative of small-molecule drugs, and cross-field and linear-field plasma jets were used for the purpose of enhancing the penetration of NaNO2 through the gelatin gel films. The permeability of gelatin gel films was quantified by measuring the NO2− concentration in water which was covered by those films. It was found that the gas flow and electric field of cold plasmas played a crucial role in the permeability enhancement of the model tissues, but the effect of gas flow was mainly confined in the surface layer, while the effect of the electric field was holistic. Those effects might be attributed to the localized squeezing of particles by gas flow and the weakening of the ion-dipole interaction by the AC electric field. The enhancement effect decreases with the increasing mass fraction of gelatin because the macromolecules of gelatin could significantly hinder the penetration of small molecules in the model tissues.

Chemical Stability of Graphene Coated Silver Substrates for Surface-Enhanced Raman Scattering

Surface enhanced Raman spectroscopy (SERS) is a novel method to sense molecular and lattice vibrations at a high sensitivity. Although nanostructured silver surface provides intense SERS signals, the silver surface is unstable under acidic environment and heated environment. Graphene, a single atomic carbon layer, has a prominent stability for chemical agents, and its honeycomb lattice completely prevents the penetration of small molecules. Here, we fabricated a SERS substrate by combining nanostructured silver surface and single-crystal monolayer graphene (G-SERS), and focused on its chemical stability. The G-SERS substrate showed SERS even in concentrated hydrochloric acid (35–37%) and heated air up to 400 °C, which is hardly obtainable by normal silver SERS substrates. The chemically stable G-SERS substrate posesses a practical and feasible application, and its high chemical stability provides a new type of SERS technique such as molecular detections at high temperatures or in extreme acidic conditions.

Abstract 5782: A novel method for high-throughput drug screening in 3D tumor organoids

Abstract 3D tumor organoids are pivotal tools to study tumor development, propagation and invasive properties (Tanner and Gottesman, 2015). They closely recapitulate many characteristics of the tumor of origin in terms of structure, heterogeneity, complexity and response to drug treatment. As such, they are considered an accurate pre-clinical model for drug discovery and development (Nyga et al, 2011; Tanner and Gottesman, 2015; Shroyer, 2016). Nevertheless, reliable methods to perform high-throughput drug screenings using tumor organoids are limited and require extensive manipulation such as sample transfer from plate to plate. In addition, thick 3D matrix supports may hinder drug penetration. Here we introduce a robust method to generate, treat and assay 3D tumor organoids in standard, uncoated 96- or 384-well plates. By plating cells around the rim of the wells we generate an ultrathin layer of Matrigel that allows penetration of small molecules and bulkier peptide drugs. Media change is easily performed from the center of the well without perturbing the organoids. After a 2-day treatment course, organoids are released by dispase treatment and assayed in the same well by performing a CellTiter-Glo assay (Promega). The method exhibits low plate-to-plate, experiment-to-experiment and operator-to-operator variability. In addition, it can be extended to patient-derived tumor organoids (PDTO) generated from primary tumors. We show how PDTO from high-grade serous ovarian carcinoma (HGSOC) specimens are generated with a success rate >95%. Given the low number of cells required and high assay sensitivity, we can seed 200 wells from 1Mio cells derived from a tumor biopsy, and test over 60 different drugs/concentrations in triplicates. Moreover, we show how drug combination studies are feasible and allow assessing treatment synergies. As an example, we combined a peptidic inhibitor of p53 aggregation, ReACp53 (Soragni et al, 2016), with carboplatin in PDTO HGSOC established from a panel of patients harboring different p53 mutations. Drug interaction studies are performed across a range of ReACp53 and carboplatin concentrations and cell viability assessed at day 2 is used to compute drug-response curves, EC50 values and combination indexes. In conclusion, we are introducing an inexpensive, robust, easily automated method to perform high-throughput drug screenings in 3D organoids and PDTO and demonstrate its utility in determining drug sensitivities and optimizing multi-drug combinations in vitro. Citation Format: Nhan Phan, Jessica Huang, David Eisenberg, Sanaz Memarzadeh, Alice Soragni. A novel method for high-throughput drug screening in 3D tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5782. doi:10.1158/1538-7445.AM2017-5782

Diffusion of Selective Penetrants in Interfacially Modified Block Copolymers from Molecular Dynamics Simulations.

To show the influence of the interface on structure and dynamics of microphase separated polymer systems, we study interfacially modified AB block copolymers with small molecule penetrants. The polymers have a random midblock or tapered midblock whose composition varies from pure A to pure B (or from pure B to pure A for an inverse taper) between two pure blocks of A and B. We perform simple coarse-grained molecular dynamics simulations of symmetric polymers that form lamellae. With normal tapering, both polymer and penetrant diffusion parallel to the lamellae increases as taper length increases. Inverse tapered polymers exist in different conformational states (e.g., stretched vs folded back and forth across the interface) with different dynamic behavior, leading to nonmonotonic trends in their diffusion. However, the local mixing of monomers (rather than polymer conformation) appears to be the most important factor in determining penetrant diffusion.

Abstract 3769: Continuous, low intensity systemic dosing maximizes the therapeutic margin of Eg5/KSP inhibition in an orthotopic model of urothelial cell carcinoma

Abstract Urothelial Cell Carcinoma (UCC) is the fifth most common cancer, but no new generation molecularly targeted therapies have been registered to treat this disease. Non muscle-invasive bladder cancer can be treated by systemic or intravesical dosing routes. However, the urothelium is one of the most formidable permeability barriers in nature, and may limit the penetration of small molecules into tumour tissue from the intravesical location. Inhibitors of mitosis, including spindle proteins such as Eg5, are attractive targets for cancer therapy, but their efficacy when dosed systemically is severely limited by bone marrow toxicities such as neutropenia and thrombocytopenia. AZ9814, a very potent and selective small molecule inhibitor of Eg5, induces apoptosis and inhibits proliferation of UCC cells in culture with a GI50 of < 1 nM. We have developed an orthotopic model of bladder cancer in the nude rat coupled with a programmable mini-pump system to continuously infuse AZ9814 into the bladder, and compared the therapeutic index of this delivery route with systemic dosing schedules using intraperitoneal injection and continuous subcutaneous infusion using an osmotic mini-pump. Continuous intravesical infusion resulted in severe bladder toxicity including hydropic degeneration and ulceration of urothelium, mucosal erosion and bilateral hydronephrosis, with limited penetration of compound into the tumour, and no significant impact on tumour pharmacodynamics and growth. In contrast, continuous low intensity systemic dosing resulted in significant tumour pharamacodynamic activity and control of tumour growth, albeit with significant but non-lethal effects on bone marrow toxicity. Daily intraperitoneal injection of AZ9814 resulted in very modest anti-tumour effects which were limited by bone marrow toxicity. It is concluded that Eg5 inhibitors have potential to treat UCC, but continuous systemic infusion is the best dosing schedule to maximize therapeutic index. Citation Format: Leigh Williams, Rebecca Ellston, Dawn Trueman, Helen Musgrove, Linette Ruston, Brian Aquila, Elizabeth Pease, Stephanie Klein, Barry R. Davies. Continuous, low intensity systemic dosing maximizes the therapeutic margin of Eg5/KSP inhibition in an orthotopic model of urothelial cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3769.

A BOILED-Egg To Predict Gastrointestinal Absorption and Brain Penetration of Small Molecules.

Apart from efficacy and toxicity, many drug development failures are imputable to poor pharmacokinetics and bioavailability. Gastrointestinal absorption and brain access are two pharmacokinetic behaviors crucial to estimate at various stages of the drug discovery processes. To this end, the Brain Or IntestinaL EstimateD permeation method (BOILED‐Egg) is proposed as an accurate predictive model that works by computing the lipophilicity and polarity of small molecules. Concomitant predictions for both brain and intestinal permeation are obtained from the same two physicochemical descriptors and straightforwardly translated into molecular design, owing to the speed, accuracy, conceptual simplicity and clear graphical output of the model. The BOILED‐Egg can be applied in a variety of settings, from the filtering of chemical libraries at the early steps of drug discovery, to the evaluation of drug candidates for development.

Enhancement of Small Molecule Delivery by Pulsed High-Intensity Focused Ultrasound: A Parameter Exploration

Chemotherapeutic drug delivery is often ineffective within solid tumors, but increasing the drug dose would result in systemic toxicity. The use of high-intensity focused ultrasound (HIFU) has the potential to enhance penetration of small molecules. However, operation parameters need to be optimized before the use of chemotherapeutic drugs in vivo and translation to clinical trials. In this study, the effects of pulsed HIFU (pHIFU) parameters (spatial-average pulse-average intensity, duty factor and pulse repetition frequency) on the penetration as well as content of small molecules were evaluated in ex vivo porcine kidneys. Specific HIFU parameters resulted in more than 40 times greater Evans blue content and 3.5 times the penetration depth compared with untreated samples. When selected parameters were applied to porcine kidneys in vivo, a 2.3-fold increase in concentration was obtained after a 2-min exposure to pHIFU. Pulsed HIFU has been found to be an effective modality to enhance both the concentration and penetration depth of small molecules in tissue using the optimized HIFU parameters. Although, performed in normal tissue, this study has the promise of translation into tumor tissue.

Vacuolization in Cytoplasm and Cell Membrane Permeability Enhancement Triggered by Micrometer-Sized Graphene Oxide.

A deep understanding of the interaction of a graphene oxide (GO) sheet with cells at the molecular level may expedite its biomedical application and predict its new functions and adverse effects. Herein we inspect the interaction between micrometer-sized GO (mGO), commonly used in biomedical research, and cells at the molecular level through a variety of techniques. A major finding is that, instead of direct cellular penetration, the mGO sheets can stimulate the cellular response by interacting with the membrane protein and the membrane. Specifically, it is illustrated that even within a short exposure time the mGO sheets can induce the formation of vacuoles in the cytosolic compartment and enhance the cell permeability. The vacuolization is only observed in the cells that strongly express aquaporin (AQP1), indicating the specific interaction of the mGO with AQP1. Moreover, inhibition of the AQP1 activity prevents the formation of vacuoles, revealing that the interaction of the mGO with AQP1 occurs most probably at the vestibule of AQP1 at the extracellular side. Additionally, though the cell permeability was enhanced, it only improves the penetration of small molecules, not for macromolecules such as proteins. These findings are potentially valuable in cancer therapy because AQPs are strongly expressed in tumor cells of different origins, particularly aggressive tumors, and it will also be beneficial for drug transport across barrier membranes.

In vitro directed evolution of alpha-hemolysin by liposome display.

We have developed a method to enable in vitro directed evolution that can be applied to membrane proteins. This method, termed liposome display, uses liposomes as compartments in which membrane proteins are synthesized and as scaffolds for membrane protein integration. Thus, the synthesized membrane proteins are displayed on the surface of the liposome and exhibit their functions. A randomly mutated DNA library of the membrane protein was generated, encapsulated in the liposomes at the single-molecule level, and used to generate a liposome library. Liposomes displaying the desired membrane protein function were selected, thus accumulating the DNA molecule encoding the desired membrane protein. We have applied this method to alpha-hemolysin, a membrane protein derived from Staphylococcus aureus. Alpha-hemolysin forms a nanopore in the membrane, which allows the penetration of small molecules. We aimed to improve this nanopore activity by using the liposome display method. Consequently, alpha-hemolysin evolved and attained a higher specific affinity for the liposome membrane. In this review, we describe the essential characteristics of liposome display and the properties of the evolved alpha-hemolysin obtained by this method.

The Impact of Hemi-brain Irradiation on Accumulation of PI3K/mTOR Inhibitors With Limited (GDC-0980) and Robust (GNE-317) Blood–Brain Barrier Penetration

Previous studies have demonstrated the physical and biochemical effects of radiation on the blood-brain barrier (BBB), which has led to a commonly held belief that radiation therapy (RT) can improve the penetration of small molecules into the brain and associated brain tumors. However, the impact of brain RT on therapeutic drug delivery has not been previously published. Here, we used hemi-brain irradiation to compare accumulation of two structurally similar PI3K/mTOR inhibitors, GNE-317 (BBB-penetrant), and GDC-0980 (BBB-impenetrant), at various time points following RT with 40 Gy in 1 or 10 fractions.

Study of secondary relaxation transitions in polysulfone and polycarbonate by a method of conformational probes

Introduction. Local molecular dynamics and secondary relaxation transitions in glassy polymers hold great practical and theoretical interest because their mechanical, thermal, dielectric, and diffusion properties, in particular, the penetration of small molecules through protective coatings, layers, and separating membranes, depend considerably on the types of molecular mobility occurring in the polymer. Local molecular dynamics in the glassy polymers polymethylmethacrylate, polyvinylacetate, polyvinyltrimethylsilane, polytrimethylsilylpropyne, and polyesterimides, which are used as gas-separating membranes, were studied earlier using an IR spectroscopic method of conformational probes [1]. Herein polysulfone (PSF) and polycarbonate (PC), which are used for membrane micro- and ultrafultration separation of mixtures of compounds [2], are investigated. Experimental. Powdered PSFs (MW = 35,000 and Mη = 16,000) and PC (MW = 64,000) (Aldrich) had glassification temperatures 463 and 423 K. The conformational probes were 1,2-dichloroethane, ClCH2–CH2Cl (DCE);