Ablative fractional CO2 laser treatment promotes wound healing phenotype in skin macrophages.

OBJECTIVES: Ablative fractional laser (AFL) treatment is a well-established method for reducing signs of skin photoaging. However, the biological mechanisms underlying AFL-induced healing responses and skin rejuvenation remain largely unknown. It is known that macrophages play an important role in orchestrating healing, normalization, and remodeling processes in skin. Macrophage phenotypes are characterized by inflammatory markers, including arginase-1 (Arg1), major histocompatibility class II molecules (MHC II), and CD206. This study aims to explore AFL's effect on macrophage phenotype by evaluating changes in inflammatory markers and the potential concurrent accumulation of Arg1 in the skin. METHODS: Mice (n = 9) received a single AFL treatment on the left side of the back skin (100 mJ/microbeam, 5% density) while the right side of the back remained untreated as control. Treated and untreated skin from each mouse were collected Day 5 posttreatment for flow cytometry and histology analysis. Flow cytometry evaluated the immune infiltration of macrophages and the expression of macrophage inflammatory markers (Arg1, MHC II, and CD206). In addition, Arg1 presence in the skin was evaluated through antibody staining of histology samples and quantification was performed using QuPath image analysis software. RESULTS: Following AFL, the number of macrophages increased 11-fold (p = 0.0053). Phenotype analysis of AFL-treated skin revealed an increase in the percentage of macrophages positive for Arg1 (p < 0.0001) and a decrease in the percentage of macrophages positive for MHC II (p < 0.0001) compared to untreated skin. No significant differences were observed in percentage of CD206-positive macrophages (p = 0.8952). Visualization of AFL-treated skin demonstrated a distinct pattern of Arg1 accumulation that correlated with the microscopic treatment zones (MTZ). Quantification of the percentage of Arg1-positive area in epidermis and dermis showed a significant increase from 3.5% ± 1.2% to 5.2% ± 1.7 (p = 0.0232) and an increase from 2.2% ± 1.2% to 9.6% ± 3.3 (p < 0.0001) in whole skin samples. CONCLUSION: AFL treatment polarizes macrophages toward a wound healing phenotype and induces Arg1 accumulation in the MTZ. We propose that the polarized wound healing macrophages are a major source for the increased Arg1 levels observed in the skin following treatment.

Laser-assisted topical delivery of vismodegib reduces hedgehog gene expression in human basal cell carcinomas in vivo.

BACKGROUND: Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs. METHODS: In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3). RESULTS: Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure. CONCLUSION: A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions.

Investigating Generation of Antibodies against the Lipid Nanoparticle Vector Following COVID-19 Vaccination with an mRNA Vaccine.

Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3-4 weeks after the first vaccination dose but before the second dose, and 2-6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.

Brain proteome profiling implicates the complement and coagulation cascade in multiple system atrophy brain pathology.

BACKGROUND: Multiple system atrophy (MSA) is a rare, progressive, neurodegenerative disorder presenting glia pathology. Still, disease etiology and pathophysiology are unknown, but neuro-inflammation and vascular disruption may be contributing factors to the disease progression. Here, we performed an ex vivo deep proteome profiling of the prefrontal cortex of MSA patients to reveal disease-relevant molecular neuropathological processes. Observations were validated in plasma and cerebrospinal fluid (CSF) of novel cross-sectional patient cohorts. METHODS: Brains from 45 MSA patients and 30 normal controls (CTRLs) were included. Brain samples were homogenized and trypsinized for peptide formation and analyzed by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Results were supplemented by western blotting, immuno-capture, tissue clearing and 3D imaging, immunohistochemistry and immunofluorescence. Subsequent measurements of glial fibrillary acid protein (GFAP) and neuro-filament light chain (NFL) levels were performed by immunoblotting in plasma of 20 MSA patients and 20 CTRLs. Finally, we performed a proteome profiling of 144 CSF samples from MSA and CTRLs, as well as other parkinsonian disorders. Data were analyzed using relevant parametric and non-parametric two-sample tests or linear regression tests followed by post hoc tests corrected for multiple testing. Additionally, high-throughput bioinformatic analyses were applied. RESULTS: We quantified more than 4,000 proteins across samples and identified 49 differentially expressed proteins with significantly different abundances in MSA patients compared with CTRLs. Pathway analyses showed enrichment of processes related to fibrinolysis and complement cascade activation. Increased fibrinogen subunit ß (FGB) protein levels were further verified, and we identified an enriched recognition of FGB by IgGs as well as intra-parenchymal accumulation around blood vessels. We corroborated blood-brain barrier leakage by a significant increase in GFAP and NFL plasma levels in MSA patients that correlated to disease severity and/or duration. Proteome profiling of CSF samples acquired during the disease course, confirmed increased total fibrinogen levels and immune-related components in the soluble fraction of MSA patients. This was also true for the other atypical parkinsonian disorders, dementia with Lewy bodies and progressive supra-nuclear palsy, but not for Parkinson's disease patients. CONCLUSION: Our results implicate activation of the fibrinolytic cascade and immune system in the brain as contributing factors in MSA associated with a more severe disease course.

Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Brain tumor vessels-a barrier for drug delivery.

Cancer treatment remains a challenge due to a high level of intra- and intertumoral heterogeneity and the rapid development of chemoresistance. In the brain, this is further hampered by the blood-brain barrier that reduces passive diffusion of drugs to a minimum. Tumors grow invasively and form new blood vessels, also in brain tissue where remodeling of pre-existing vasculature is substantial. The cancer-associated vessels in the brain are considered leaky and thus could facilitate the transport of chemotherapeutic agents. Yet, brain tumors are extremely difficult to treat, and, in this review, we will address how different aspects of the vasculature in brain tumors contribute to this.

Simultaneous Cross-Linking and Cross-Polymerization of Enzyme Responsive Polyethylene Glycol Nanogels in Confined Aqueous Droplets for Reduction of Low-Density Lipoprotein Oxidation.

A key initiating step in atherosclerosis is the accumulation and retention of apolipoprotein B complexing lipoproteins within the artery walls. In this work, we address this exact initiating mechanism of atherosclerosis, which results from the oxidation of low-density lipoproteins (oxLDL) using therapeutic nanogels. We present the development of biocompatible polyethylene glycol (PEG) cross-linked nanogels formed from a single simultaneous cross-linking and co-polymerization step in water without the requirement for an organic solvent, high temperature, or shear stress. The nanogel synthesis also incorporates in situ noncovalent electrostatically driven template polymerization around an innate anti-inflammatory and anti-oxidizing paraoxonase-1 (PON-1) enzyme payload-the release of which is triggered because of matrix metalloproteinase responsive elements instilled in the PEG cross-linker monomer. The results obtained demonstrate the potential of triggered release of the PON-1 enzyme and its efficacy against the production of ox-LDL, and therefore a reduction in macrophage foam cell and reactive oxygen species formation.

Unique Calibrators Derived from Fluorescence-Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations.

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.

The Composition of Reconstituted High-Density Lipoproteins (rHDL) Dictates the Degree of rHDL Cargo- and Size-Remodeling via Direct Interactions with Endogenous Lipoproteins.

The application of reconstituted high-density lipoproteins (rHDL) as a drug-carrier has during the past decade been established as a promising approach for effective receptor-mediated drug delivery, and its ability to target tumors has recently been confirmed in a clinical trial. The rHDL mimics the endogenous HDL, which is known to be highly dynamic and undergo extensive enzyme-mediated remodulations. Hence, to reveal the physiological rHDL stability, a thorough characterization of the dynamics of rHDL in biologically relevant environments is needed. We employ a size-exclusion chromatography (SEC) method to evaluate the dynamics of discoidal rHDL in fetal bovine serum (FBS), where we track both the rHDL lipids (by the fluorescence from lipid-conjugated fluorophores) and apoA-I (by human apoA-I ELISA). We show by using lipoprotein depleted FBS and isolated lipoproteins that rHDL lipids can be transferred to endogenous lipoproteins via direct interactions in a nonenzymatic process, resulting in rHDL compositional- and size-remodeling. This type of dynamics could lead to misinterpretations of fluorescence-based rHDL uptake studies due to desorption of labile lipophilic fluorophores or off-target side effects due to desorption of incorporated drugs. Importantly, we show how the degree of rHDL remodeling can be controlled by the compositional design of the rHDL. Understanding the correlation between the molecular properties of the rHDL constituents and their collective dynamics is essential for improving the rHDL-based drug delivery platform. Taken together, our work highlights the need to carefully consider the compositional design of rHDL and test its stability in a biological relevant environment, when developing rHDL for drug delivery purposes.

Protein Transduction Domain Mimics Facilitate Rapid Antigen Delivery into Monocytes.

Delivering peptides and proteins with intracellular function represents a promising avenue for therapeutics, but remains a challenge due to the selective permeability of the plasma membrane. The successful delivery of cytosolically active proteins would enable many opportunities, including improved vaccine development through major histocompatibility complex (MHC) class I antigen display. Extended research using cell-penetrating peptides (CPPs) has aimed to facilitate intracellular delivery of exogenous proteins with some success. A new class of polymer-based mimics termed protein transduction domain mimics (PTDMs), which maintain the positive charge and amphiphilic nature displayed by many CPPs, was developed using a poly-norbornene-based backbone. Herein, we use a previously characterized PTDM to investigate delivery of the model antigen SIINFEKL into leukocytes. Peptide delivery into over 90% of CD14+ monocytes was detected in less than 15 min with nominal inflammatory cytokine response and high cell viability. The co-delivery of a TLR9 agonist and antigen using the PTDM into antigen-presenting cells in vitro showed presentation of SIINFEKL in association with MHC class I molecules, in addition to upregulation of classical differentiation markers revealing the ability of the PTDM to successfully deliver cargo intracellularly and show application in the field of immunotherapy.

Topical delivery of vismodegib using ablative fractional laser and micro-emulsion formulation in vitro.

BACKGROUND AND OBJECTIVE: Hedgehog inhibitors such as vismodegib are targeted drugs widely used for the treatment of basal cell carcinomas; however, their use is significantly limited by frequent systemic side effects due to oral administration route. We aim to use ablative fractional laser (AFL) to enable the topical delivery of vismodegib to relevant dermal depths. MATERIALS AND METHODS: Pig skin was treated in vitro with a fractional 10,600 nm CO2 laser at 0 or 80 mJ/microbeam and exposed to vismodegib (6.4 mmol/L) in Franz-diffusion cells for 0.5, 4, and 24 hours (n = 54 samples), either formulated in a micro-emulsion composed of soybean oil and Tween 80 or dissolved in ethanol as vehicle control. Vismodegib biodistribution was studied at specific skin depths from 0 to 1,800 µm (incremental steps of 300 µm) by mass spectrometry. RESULTS: Combination of AFL and vismodegib emulsion substantially enhanced the delivery of drug into the skin. Emulsion formulation alone yielded higher vismodegib skin concentrations compared to vehicle control in superficial and mid-dermis (0-900 µm, P = 0.002-0.015). The over-all highest concentration found (554.5 µmol/L) was reached at 24 hours in superficial (0-300 µm) AFL exposed skin, 7.6-fold higher than vehicle control (P = 0.002) and 9.7-101.6 fold higher than previously reported steady-state plasma concentrations in patients treated with oral vismodegib (5.5-56.9 µmol/L). Compared to intact skin, AFL exposure significantly increased skin concentrations of vismodegib even in deep skin layers (24 h, 900-1,800 µm, emulsion: 8.7-74.3 µmol/L vs. 0.0-0.0 µmol/L, P = 0.004-0.048; vehicle control: 23.7-50.6 µmol/L vs. 0.0-1.6 µmol/L, P = 0.002). The total delivery of vismodegib-emulsion into mid-deep dermal skin layers from 600 to 1,800 µm was for AFL exposed skin 8.2 fold higher than intact skin. Also, delivery of emulsion vismodegib by AFL was time-dependent as seen by the continuous increase in concentrations found over time, with highest uptake detected after 24 hours (4-24 hours, 0-900 µm, P = 0.002-0.004). CONCLUSION: AFL enhances topical delivery of micro-emulsion formulated vismodegib, reaching concentrations similar to or above plasma concentrations previously reported in patients receiving oral vismodegib. Lasers Surg. Med. 51:79-87, 2019. © 2018 Wiley Periodicals, Inc.

How To Characterize Individual Nanosize Liposomes with Simple Self-Calibrating Fluorescence Microscopy.

Nanosize lipid vesicles are used extensively at the interface between nanotechnology and biology, e.g., as containers for chemical reactions at minute concentrations and vehicles for targeted delivery of pharmaceuticals. Typically, vesicle samples are heterogeneous as regards vesicle size and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, separately. A vesicle then images as two spots, one in each color channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles. They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Nonideal vesicles in the same images were characterized by how their four data violate the calibrated relationship established for ideal vesicles. In this way, our method yields size, shape, lamellarity, and encapsulation efficiency of each imaged vesicle. Applying this procedure to extruded samples of vesicles, we found that, contrary to common assumptions, only a fraction of vesicles are ideal.

PET imaging with copper-64 as a tool for real-time in vivo investigations of the necessity for cross-linking of polymeric micelles in nanomedicine.

Polymeric micelles in nanomedicine are often cross-linked to prevent disintegration in vivo. This typically requires clinically problematic chemicals or laborious procedures. In addition, cross-linking may interfere with advanced release strategies. Despite this, it is often not investigated whether cross-linking is necessary for efficient drug delivery. We used positron emission tomography (PET) imaging with 64 Cu to demonstrate general methodology for real-time in vivo investigations of micelle stability. Triblock copolymers with 4-methylcoumarin cores of ABC-type (PEG-PHEMA-PCMA) were functionalized in the handle region (PHEMA) with CB-TE2A chelators. Polymeric micelles were formed by dialysis and one half was core cross-linked (CL) by UV light and the other half was not (nonCL). Both CL and nonCL were radiolabeled with 64 Cu and compared in vivo in tumor-bearing mice, with free 64 Cu as control. Accumulation in relevant organs was quantified by region of interest analysis on PET images and ex vivo counting. It was observed that CL and nonCL showed limited differences in biodistribution from each other, whereas both differed markedly from control (free 64 Cu). This demonstrated that 4-methylcoumarin core micelles may form micelles that are stable in circulation even without cross-linking. The methodology presented here where individual unimers are radiolabeled is applicable to a wide range of polymeric micelle types.

Affinity Induced Surface Functionalization of Liposomes Using Cu-Free Click Chemistry.

Functionalization of nanoparticles is a key element for improving specificity of drug delivery systems toward diseased tissue or cells. In the current study we report a highly efficient and chemoselective method for post-functionalization of liposomes with biomacromolecules, which equally well can be used for functionalization of other nanoparticles or solid surfaces. The method exploits a synergistic effect of having both affinity and covalent anchoring tags on the surface of the liposome. This was achieved by synthesizing a peptide linker system that uses Cu-free strain-promoted click chemistry in combination with histidine affinity tags. The investigation of post-functionalization of PEGylated liposomes was performed with a cyclic RGDfE peptide. By exploring both affinity and covalent tags a 98 ± 2.0% coupling efficiency was achieved, even a diluted system showed a coupling efficiency of 87 ± 0.2%. The reaction kinetics and overall yield were quantified by HPLC. The results presented here open new possibilities for constructing complex nanostructures and functionalized surfaces.

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.

Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs.

In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.

Single-vesicle detection and analysis of peptide-induced membrane permeabilization.

The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.

Synthesis of Cross-Linked Polymeric Micelle pH Nanosensors: An Investigation of Design Flexibility.

The design flexibility that polymeric micelles offer in the fabrication of optical nanosensors for ratiometric pH measurements is investigated. pH nanosensors based on polymeric micelles are synthesized either by a mixed-micellization approach or by a postmicelle modification strategy. In the mixed-micellization approach, self-assembly of functionalized unimers followed by shell cross-linking by copper-catalyzed azide-alkyne cycloaddition (CuAAC) results in stabilized cRGD-functionalized micelle pH nanosensors. In the postmicelle modification strategy, simultaneous cross-linking and fluorophore conjugation at the micelle shell using CuAAC results in a stabilized micelle pH nanosensor. Compared to the postmicelle modification strategy, the mixed-micellization approach increases the control of the overall composition of the nanosensors. Both approaches provide stable nanosensors with similar pKa profiles and thereby nanosensors with similar pH sensitivity.

In vivo toxicity of cationic micelles and liposomes.

This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR: This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Positron emission tomography based analysis of long-circulating cross-linked triblock polymeric micelles in a U87MG mouse xenograft model and comparison of DOTA and CB-TE2A as chelators of copper-64.

Copolymers of ABC-type (PEG-PHEMA-PCMA) architecture were prepared by atom transfer radical polymerization and formulated as micelles with functionalizable primary alcohols in the shell-region (PHEMA-block) to which the metal-ion chelators DOTA or CB-TE2A were conjugated. Using this micelle system we compared the in vivo stabilities of DOTA and CB-TE2A as chelators of (64)Cu in micelle nanoparticles. The coumarin polymer (PCMA-block) micelle core was cross-linked by UV irradiation at 2 W/cm(2) for 30 min. The cross-linked micelles were labeled with (64)Cu at room temperature for 2 h (DOTA) or 80 °C for 3 h (CB-TE2A), giving labeling efficiencies of 60-76% (DOTA) and 40-47% (CB-TE2A). (64)Cu-micelles were injected into tumor-bearing mice (8 mg/kg) and PET/CT scans were carried out at 1, 22, and 46 h postinjection. The micelles showed good blood stability (T1/2: 20-26 h) and tumor uptake that was comparable with other nanoparticle systems. The DOTA micelles showed a biodistribution similar to the CB-TE2A micelles and the tumor uptake was comparable for both micelle types at 1 h (1.9% ID/g) and 22 h (3.9% ID/g) but diverged at 46 h with 3.6% ID/g (DOTA) and 4.9% ID/g (CB-TE2A). On the basis of our data, we conclude that cross-linked PEG-PHEMA-PCMA micelles have long circulating properties resulting in tumor accumulation and that DOTA and CB-TE2A (64)Cu-chelates show similar in vivo stability for the studied micelle system.