PEG Lipid Research Articles

Size-dependent biodistribution of carbon nanohorns in vivo

Carbon nanotubules, such as nanotubes and nanohorns, are potentially useful as drug delivery or hyperthermia agents for cancer therapy. However, the biokinetics of variously sized nanocarbons are important for their medical application and risk assessment. To examine the time course of the biodistribution of carbon nanohorns (CNHs) in mice, CNH aggregates of 100nm (L-CNHs) or CNHs of 30–50nm (S-CNHs) were dispersed with lipid polyethylene glycol and administered to mice through tail vein injection. Histological observation revealed that S-CNHs accumulated more slowly than did L-CNHs in the liver and spleen. The accumulation of L- and S-CNHs in spleen reached saturation within 1 and 48h, respectively, and the accumulation in liver reached saturation within 48h and >7days, respectively. CNHs did not accumulate appreciably in the lung, skin, or kidney. Histologic, hematologic, and immunologic (IL-6, TNF-α, and IFN-γ) tests did not reveal obvious toxicologic lesions at any time point. From the Clinical EditorIn this study the biodistribution and accumulation characteristics of small and large carbon nanohorns were characterized in mice. Data demonstrate slower accumulation of small carbon nanohorns in liver and spleen, no accumulation in skin, lung, or kidney, and no obvious hematologic or immunologic toxicity.

【原著】 ポストインサーション法によって調製したPEG修飾リポソームの表面物性に関する研究

Post-insertion method is now widely used to modify liposomes with poly(ethylene glycol) (PEG) lipids. In this study, we examined the physicochemical properties of surface layers of distearoylphosphatidylethanolamine- PEG2000-modified liposomes composed of distearoylphosphatidylcoline, distearoylphosphatidylglycerol and cholesterol prepared in post-insertion method. Zeta potential measurements showed that the estimated aqueous layer thickness of PEG liposomes increases with increasing concentrations of PEG lipids added. Size exclusion chromatography demonstrated that the incorporation efficiency of PEG lipids into liposomes gradually decreases with increasing addition amount of PEG lipids. Quenching experiments of fluorescence-labeled PEG lipids confirmed that almost all PEG lipids distribute at the outermost layer of liposomes in the post-insertion preparation whereas PEG lipids distribute equally at inner and outer layers of liposomes in the conventional pre-mixed method. By correcting the incorporation efficiency and the outermost distribution of PEG lipids in liposomes, we found that the dependency of increases in aqueous layer thickness of PEG liposomes upon the outermost content of PEG lipids is similar in the both post-insertion and pre-mixed methods. Fluorescence resonance energy transfer measurements suggested that the increasing behavior of aqueous layer thickness is closely correlated with the conformational transition of PEG chains at the liposome surface. These results provide comparative information of the surface properties of PEG-modified liposomes prepared by the post-insertion and pre-mixed methods.

Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles

Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.

A photo-triggerable drug carrier based on cleavage of PEG lipids by photosensitiser-generated reactive singlet oxygen

To circumvent the limitations of polyethylene glycol (PEG) modified carriers, a photo-triggerable liposome was prepared which was modified by cholesterol derivatives via a cleavable vinyl ether linkage so that the PEGylated coating can be efficiently removed by a photoactivated fullerene. After the photocleavage of the PEG moiety, the intracellular uptake of the photo-triggerable liposome improved.

Electrochemical and PM-IRRAS studies of floating lipid bilayers assembled at the Au(111) electrode pre-modified with a hydrophilic monolayer

A floating bilayer lipid membrane (fBLM) assembled on a 1-thio-β-D-glucose (β-Tg)-modified Au(111) surface was engineered to mimic the environment of a biological cell membrane. This bilayer consists of a mixture of the phospholipid (DMPC), polyethylene glycol lipid (DMPE-PEG350) and cholesterol. Prior to membrane adsorption, a monolayer of β-Tg was first self-assembled at the Au(111) surface to increase the hydrophilicity of the electrode surface. The asymmetric bilayer was then deposited onto the hydrophilic support using a combination of Langmuir–Blodgett and Langmuir–Schaefer techniques. The inner leaflet of the bilayer was composed of DMPC, DMPE-PEG350 and cholesterol, while the outer leaflet consisted of DMPC and cholesterol. The hydrophilic PEG spacer subunit of the DMPE-PEG350 molecule promotes the formation of a water-rich domain, which separates the bilayer from the β-Tg modified surface. The Langmuir compression isotherms were recorded to examine the miscibility of these mixed monolayers at the air-solution interface of the Langmuir trough. Differential capacitance measurements showed that this fBLM was remained stable on the surface of the modified-gold electrode for potentials ranging from −0.40 to 0.45V versus Ag/AgCl and achieved a minimum capacitance of ∼3.0μFcm−2. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to obtain information regarding the conformation and orientation of the acyl chains and measure the degree of hydration of the phospholipid polar head groups within the film. These spectra demonstrated that the acyl chains of the lipids assume a small angle with respect to surface normal and that the phospho-glycerol groups are well-hydrated. The IR spectra of the PEG subunits demonstrated that the spacer unit adopts an amorphous, disordered conformation, which favors the hydration of the inner layer. This work demonstrates that the fBLM, composed of DMPE-PEG350 molecules, forms a water-rich region that separates the bilayer from the metal surface and that these new biomimetic systems are promising for future investigations involving transmembrane proteins.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Abstract 2888: Lipid nanoparticle-mediated delivery of siRNA to extrahepatic tumors

Abstract While lipid nanoparticles (LNPs) proved to be effective in delivering siRNA to the liver and hepatocellular carcinoma (HCC) via systemic administration, they showed inferior or no efficacy in the extrahepatic tumor models. To understand the underlying mechanism for LNP activity in delivering siRNA to extrahepatic tumors, we determined siRNA biodistribution and gene-silencing activity of the LNP-siRNA nanoparticles containing different percentages of a polyethylene glycol (PEG) lipid, distinct PEG lipids with a short (C-14) or long (C-18) alkyl chain, or an identical composition with different particle sizes in mouse subcutaneous tumor xenograft models derived from either human HCC (Hep3B) or colon cancer (HT29 or HCT116) cells. We show that the LNPs containing C-18 DSA PEG exhibited a higher retention of siRNA in both Hep3B and HT29 tumors than those with C-14 DMA PEG following an intravenous administration. Intriguingly, LNPs containing 2% of DSA or DMA PEG resulted in similar levels (∼50%) of target knockdown in Hep3B tumors while those containing 6% of DSA or DMA PEG caused no target silencing. Also, none of these LNPs reduced target expression in HT29 tumors despite the retention of siRNA in tumor tissues. When LNPs with an identical composition (2% DMG PEG) but different particle sizes (80 nm vs. 120 nm) were tested, we observed a comparable and moderate suppression of target expression in Hep3B tumors, but no target knockdown in HCT116 tumors. This is despite the fact that the expression of low density lipid receptor, which is shown to be involved in mediating LNP efficacy, is detected in HT29 and HCT116 tumors. Finally, intratumor injection of LNPs only induced minimal target silencing in subcutaneous tumors while provoking significant target knockdown in the liver. Together, enhanced tumor retention of siRNA might not be readily translated to target silencing activity in tumors. Other factors such as the efficiency of transfecting tumor cells and the passive targeting effect through opsonization could impact on LNP efficacy in extrahepatic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2888. doi:1538-7445.AM2012-2888

Abstract 2896: Nanoparticle co-delivery of RNAi and chemotherapy for the treatment of drug-resistant cancers

Abstract The emergence of drug resistance has been a critical barrier for effective cancer chemotherapy. Recently, RNA interference (RNAi) has shown promise in suppressing the over-expression of drug resistance-associated proteins such as P-glycoprotein and Prohibitin1, and in treating multidrug resistant cancers through combination with chemotherapy. Nevertheless, the safe and effective delivery of RNAi therapeutics (e.g., siRNA and miRNA) to target cells remains a major hurdle for their clinical applications, and the co-delivery of RNAi and chemotherapeutic agents in a controlled fashion requires specific delivery vehicles. Herein, we present a novel nanoparticle (NP) platform to tackle the challenges associated with the delivery of siRNA and chemotherapeutic drugs, using biodegradable and biocompatible polymers and lipids. The hybrid lipid-polymer NP has a differentially charged hollow core/shell nanostructure which provides the delivery system with three distinct functional features: (i) a positively charged inner hollow core for dense loading of siRNA; (ii) a middle hydrophobic polymer layer for the encapsulation of anti-cancer drugs (e.g., taxanes) and for the controlled release of both siRNA and drug; and (iii) a relatively neutral lipid-polyethylene glycol (PEG) surface to keep the NP stable and prolong its systematic circulation. Moreover, such hybrid NPs can be formulated in a simple and robust way that could facilitate future scale-up. By screening various factors, such as inner cationic lipids, outer PEG chain length, and formulation parameters, the hybrid lipid-polymer NPs show excellent in vitro knockdown efficacy at low doses of siRNA, with greater than 80% luciferase silencing at an siRNA dose of 0.4 pmol (4.0 nM) and ∼ 95% silencing at a dose of 2.0 pmol (20 nM). These NPs can also efficiently reduce the expression of drug resistance-associated proteins (e.g., MDR1 and Prohibitin1), as evidenced by qPCR. Cellular cytotoxicity experiments further confirm that this NP co-delivery strategy can drastically improve the sensitivity of drug-resistant cells to chemotherapy. In addition, these hybrid NPs demonstrate promising in vivo results for co-delivering siRNA and chemotherapeutic drugs to treat drug-resistant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2896. doi:1538-7445.AM2012-2896

Effect of the Anchor in Polyethylene Glycol-Lipids on the Transfection Activity of PEGylated Cationic Liposomes Encapsulating DNA

The use of polyethylene glycol (PEG)-modified lipids (PEG-lipids) as a component of cationic liposomes impairs the cytoplasmic delivery of the encapsulated cargos by reducing endosomal escape. While this results in a loss of gene expression of encapsulated plasmid DNA, PEG-modification is useful in that it permits the formation of small, stabilized particles. In the present study, the dilemma associated with the use of PEG was overcome by modifying liposomes with stearylated INF7 (STR-INF7), a membrane fusion-independent destabilizer of endosomes, and substituting hydrophobic lipid-anchors in the PEG-lipid. The cationic liposomes modified with a series of PEG-lipids showed a drastically impaired transgene expression. However, the incorporation of STR-INF7 recovered the gene expression, and this was found to be mainly dependent on the type of PEG lipid-anchor used. Of note, the fold increase in transfection activity was highest in cholesterol-anchored PEG (>100-fold), whose enhanced endosomal escape was followed by imaging techniques. These data suggest that the structure of lipid-anchors in PEG affects the action of the peptides for inducing of endosomal escape.

Membrane Criticality Enriches Components at Sites of Adhesion

Giant plasma membrane vesicles isolated from living cells undergo large and dynamic critical fluctuations near room temperature. These results have led us to hypothesize that fluctuations persist to growth temperatures of 37°C. In this study, we demonstrate that criticality can greatly impact the organization of components at sites of adhesion between vesicles and a planar supported membrane. Adhesion is accomplished through biotin- streptavidin binding by either directly incorporating trace quantities of biotin-tagged lipids, or indirectly using a biotin conjugated Cholera Toxin B subunit that binds to the ganglioside GM1 incorporated into vesicles. Consistent with previous studies, we observe finite adhesion domains rich in the adhesion molecule (streptavidin) when a PEG lipid is incorporated into vesicles. When vesicle membranes contain coexisting liquid phases, the presence of the adhesion domain affects the localization of phase separated domains based on the partitioning of the adhesion molecules. For example, we observe enrichment of the disordered phase marker DiIC12 at the adhesion domain when the adhesion molecule is a lipid that prefers liquid-disordered phase regions. When vesicles have critical membrane compositions, DiI remains enriched at sites of adhesion at temperatures >15°C above the transition temperature. This occurs even though DiI is not directly involved in adhesion. Instead, DiI partitioning occurs likely because adhesion molecules gather fluctuations that are rich in the lipid probe. At these same temperatures, free-floating vesicles appear uniform on the micron-scale. We also observe DiI enrichment or depletion at the adhesion domain when giant plasma membrane vesicles are adhered to a supported bilayer at 37°C. This occurs even though measured critical temperatures are close to room temperature. Our results demonstrate that membrane criticality can significantly alter the organization of membrane components even when critical temperatures are well below growth temperatures.

The Effect of Formation Pathway on the Structure and Stability of PEGylated Lipoplexes at Physiological Conditions: Implications for Gene Delivery

Cationic liposome - DNA (CL-DNA) complexes (or lipoplexes) are among the most promising materials for delivery of genes into mammalian cells for gene therapy applications. These complexes are found to remain stable in monovalent salt solutions until ca. 500 mM. Long term circulation in blood requires a repulsive polymer coat to prevent removal by the immune system. A viable way of obtaining coated CL-DNA particles is incorporation of polyethylene glycol lipids (PEG-lipids) before mixing with DNA (Ewert et al. Topics Curr. Chem. 2010, 296, 191-226). Here we show that pegylated complex stability in salt depends on the preparation method of the particles. CL-DNA particles with 5 mol% of PEG 2000 form stable complexes with well defined lamellar peaks in water. The structure remains stable after addition of a 150 mM NaCl or DMEM medium (both with ionic strength near physiological conditions). Conversely, if complexes are initially formed in salt or DMEM medium, the structure is less stable with small-angle X-ray scattering (SAXS) showing the coexistence of narrow and broad lamellar peaks. If the PEG amount in the bilayers is increased to 10%, the particles formed in water continue to be well defined, but the ones formed in salt and DMEM exhibit small domain sizes as revealed in SAXS experiments. Cryo-TEM images also show onion-like structures with very few layers. Since pegylated CL-DNAs are widely used in gene therapy applications, these results are of significance since it is reasonable to expect that the degree of compactness of these particles (which depends on the preparation method) will strongly affect complex stability and transfection efficiency.Funded by: NIH GM59288. BFBS acknowledges funding from the European Community under a Marie Curie IOF grant (n252701).

Engineering liposomes as detection reagents for CD4+ T-cells

Liposomes are often used for targeted and controlled delivery of sensitive pharmaceutical compounds. They may be synthesized encapsulating drugs within their aqueous cores and their surfaces may be functionalized with antibodies and polyethylene glycol (PEG) for site-specific delivery and increased circulation time, respectively. Here we investigated the use of liposomes for cell detection in clinical diagnostics, relying on a specific biorecognition event rather than non-specific fusion with cell membranes. Streptavidin-conjugated sulforhodamine B-encapsulating liposomes were used to provide fluorescence signal amplification in a microtiter plate-based immunoassay, relying on sandwich complex formation of CD4+ T-cells between anti-CD3 or anti-CD45 antibodies and biotinylated anti-CD4+ detection antibodies. Liposomes were engineered to avoid fusion with T-cells, non-specific binding and self-aggregation, as well as promoting specific binding, encapsulation efficiency and stability. Increasing the mol% coverage of the carboxylated lipid N-glutaryl-DPPE increased the streptavidin coupling and dye encapsulation efficiency. However, increasing amounts of this lipid also increased non-specific binding to T-cells and monocytes. Thus, PEG–lipid conjugates were included in the formulation with varied coverage (0.5–3.0 mol% of total lipid) and PEG chain length (MW 350–5000). Increases in both parameters increased the liposomes' zeta potential and decreased non-specific binding, presumably due to a shielding effect on the negative charge. The optimal formulation was able to detect as few as 30 cells per μL from negatively isolated cell preparations from human blood samples. The assay responded with excellent reproducibility and discrimination between at risk and normal CD4+ cell counts over the CD4+ cell concentration range expected, which makes this method amenable to monitoring cell concentrations in those with other immunosuppressive diseases or currently receiving anti-retroviral therapy. The successful engineering of liposomes as diagnostic tools for immune cell detection expands their use to whole cell-based studies for cancer research and clinical diagnostics.

Effects of a PEG additive on the biomolecular interactions of self-assembled dendron nanostructures

The ability of self-assembling multivalent DNA-binding dendrons to interact with biological targets is modified by co-assembly with two novel low-molecular-weight cholesterol-functionalised PEG units, one based on triethylene glycol (Chol-PEG-3) and one on an octaethylene glycol (Chol-PEG-8). The addition of either PEG lipid affected the co-assembled nanostructure surface charge and size in different ways depending on the structure of the self-assembling DNA-binding dendron. Co-assembly with Chol-PEG-8 enhanced DNA binding, while Chol-PEG-3 inhibited it. Insertion of Chol-PEG-8 into the aggregates modified their ability to cross a model mucus layer, the details of which can be understood in terms of a balance between the mucoadhesivity due to the surface charge of the nanoscale aggregates and that due to the PEG groups. This study demonstrates that the interaction of nanoscale assemblies with biological systems depends on a number of different factors in a sometimes unpredictable way. Given how simply multiple building blocks can be combined by self-assembly, we conclude that self-assembled multivalent systems have great potential for optimisation to maximise their biological and clinical activity.

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: Distearoylphosphatidylethanolamine (DSPE)-PEG2000 was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG1100 was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG2000 or sterol-PEG1100 into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG1100 in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.

Paclitaxel loaded PEG 5000–DSPE micelles as pulmonary delivery platform: Formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation

The objective of the present study was to evaluate the potential of paclitaxel loaded micelles fabricated from PEG 5000–DSPE as a sustained release system following pulmonary delivery. PEG 5000–DSPE micelles containing paclitaxel were prepared by solvent evaporation technique followed by investigation of in vitro release of paclitaxel in lung simulated fluid. Tissue distribution and plasma pharmacokinetics of the PEG–lipid micelles after intratracheal and intravenous administrations were investigated in addition to intratracheally administered taxol. Finally, toxicological profile of PEG 5000–DSPE was investigated. Paclitaxel was successfully formulated in PEG–lipid micelles with encapsulation efficiency of 95%. The PEG–lipid micelles exhibited a sustained release behavior in the simulated lung fluid. Intratracheally administered polymeric micellar paclitaxel showed highest accumulation of paclitaxel in the lungs with AUC 0–12 in lungs being 45-fold higher than intravenously administered formulation and 3-fold higher than intratracheally delivered taxol. Paclitaxel concentration in other non-targeted tissues and plasma were significantly lower as compared to other groups. Furthermore, toxicity studies showed no significant increase in levels of lung injury markers in PEG 5000–DSPE treated group as compared to saline-treated group. PEG 5000–DSPE micelles delivered intratracheally were able to sustain highest paclitaxel concentrations in lungs for long periods of time, thus apprehending their suitability as pulmonary drug carriers.

Biofunctionalization of Anisotropic Nanocrystalline Semiconductor–Magnetic Heterostructures

Asymmetric binary nanocrystals (BNCs) formed by a spherical γ-Fe(2)O(3) magnetic domain epitaxially grown onto a lateral facet of a rodlike anatase TiO(2) nanorod have been functionalized with PEG-terminated phospholipids, resulting in a micellar system that enables the BNC dispersion in aqueous solution. The further processability of the obtained water-soluble BNC including PEG lipid micelles and their use in bioconjugation experiments has been successfully demonstrated by covalently binding to bovine serum albumin (BSA). The whole process has also been preliminarily performed on spherical iron oxide nanocrystals (NCs) and TiO(2) nanorods (NRs), which form single structural units in the heterostructures. Each step has been thoroughly monitored by using optical, structural, and electrophoretic techniques. In addition, an investigation of the magnetic behavior of the iron oxide NCs and BNCs, before and after incorporation into PEG lipid micelles and subsequently bioconjugation, has been carried out, revealing that the magnetic characteristics are mostly retained. The proposed approach to achieving water-soluble anisotropic BNCs and their bioconjugates has a large potential in catalysis and biomedicine and offers key functional building blocks for biosensor applications.

Incorporating functionalized polyethylene glycol lipids into reprecipitated conjugated polymernanoparticles for bioconjugation and targeted labeling of cells

We report a simple and rapid method to prepare extremely bright, functionalized, stable, and biocompatible conjugated polymer nanoparticles incorporating functionalized polyethylene glycol (PEG) lipids by reprecipitation. These nanoparticles retain the fundamental spectroscopic properties of conjugated polymer nanoparticles prepared without PEG lipid, but demonstrate greater hydrophilicity and quantum yield compared to unmodified conjugated polymer nanoparticles. The sizes of these nanoparticles, as determined by TEM, were 21-26 nm. Notably, these nanoparticles were prepared with several PEG lipid functional end groups, including biotin and carboxy moieties that can be easily conjugated to biomolecules. We have demonstrated the availability of these end groups for functionalization using the interaction of biotin PEG lipid conjugated polymer nanoparticles with streptavidin. Biotinylated PEG lipid conjugated polymer nanoparticles bound streptavidin-linked magnetic beads, while carboxy and methoxy PEG lipid modified nanoparticles did not. Similarly, biotinylated PEG lipid conjugated polymer nanoparticles bound streptavidin-coated glass slides and could be visualized as diffraction-limited spots, while nanoparticles without PEG lipid or with non-biotin PEG lipid end groups were not bound. To demonstrate that nanoparticle functionalization could be used for targeted labelling of specific cellular proteins, biotinylated PEG lipid conjugated polymer nanoparticles were bound to biotinylated anti-CD16/32 antibodies on J774A.1 cell surface receptors, using streptavidin as a linker. This work represents the first demonstration of targeted delivery of conjugated polymer nanoparticles and demonstrates the utility of these new nanoparticles for fluorescence based imaging and sensing.

SiRNA containing nanoparticles: stability of encapsulation and particle size

A large effort is currently put into the development of nano-scaled carrier systems that can guide siRNA molecules to their target cells after intravenous injection. One of the main issues in this research is the integrity of the siRNA containing nanoparticles in the blood stream. The integrity of the nanoparticles comprises both the particle size and the stable encapsulation of siRNA. Techniques currently available for studying the disassembly and size distribution of siRNA containing nanoparticles are time-consuming and incompatible with biological fluids. We initially developed a fluorescence fluctuation spectroscopy (FFS) based method which allows us to monitor the integrity of siRNA-carrier complexes in less than one minute in complex biological media and at very low siRNA concentrations. Second, while the size distribution of the complexes can be easily measured in a clear dispersion by dynamic light scattering or electron microscopy, it cannot be measured in more complex biological media such as plasma or whole blood, which contain several different interfering components. To address this issue, we have developed a novel technique, based on single particle tracking (SPT) microscopy, for studying the size distribution (and aggregation) of nanoscopic drug complexes in biological fluids. For stabilization of the particle size of cationic lipid based nanoparticles, inclusion of lipids conjugated with PEG is widely used to sterically hinder aggregate formation. We have demonstrated that in order to obtain remaining siRNA complexation to the cationic liposomes, effective encapsulation inside the liposome, or in between lipid multilayers is required, since siRNA electrostatically bound to the outer side of the liposomes is quickly pushed away by the ubiquitous albumin molecules in blood which leads to siRNA degradation and loss of effectiveness. Formation of siRNA protecting multilayers is hindered by inclusion of PEG-lipids, a hurdle that needs to be overcome either by post-insertion of the PEG-lipid into multilayer containing siRNA-liposome complexes, or by efficient encapsulation of the siRNA inside the aqueous core of the PEGylated liposome. Size stabilization in buffer can be easily achieved by inclusion of minor percentages (not, vert, similar1%) of PEG-lipids. In whole blood however, we demonstrate that much higher percentages of PEG–lipids (5–10%) are required to achieve size stabilization. This requirement has not been previously considered because of the lack of a suitable technique to study the aggregation phenomena in whole blood. In our work we demonstrate that assaying the physicochemical properties of siRNA encapsulating nanoparticles should always be carried out in the biological media they are designed to be employed in. Two novel microscopy based methods were developed that enable such characterization in biological fluids such as serum, plasma or even whole blood.

Co-immobilization of urokinase and thrombomodulin on islet surfaces by poly(ethylene glycol)-conjugated phospholipid

Transplantation of islets of Langerhans is a promising method for treating patients with insulin-dependent diabetes mellitus. The major obstacle in clinical settings is early graft loss due to inflammation triggered by blood coagulation and complement activation on the surface of the islets after intraportal transplantation. We propose a versatile method for modifying the surface of islets with the fibrinolytic enzyme urokinase and the soluble domain of the anticoagulant enzyme thrombomodulin. The surfaces of islets were modified with a poly(ethylene glycol)–phospholipid conjugate bearing a maleimide group (Mal–PEG–lipid; PEG MW=5000kDa). The Mal–PEG–lipid anchored to the cell membranes of islets, resulting in the presentation of functional maleimide groups on the islet surface. The surface was further treated with thiolated urokinase and thrombomodulin that conjugated by thiol/maleimide bonding. No practical islet volume increase was observed after surface modification, and the modifications did not impair insulin release in response to glucose stimulation. Furthermore, the activity of the immobilized urokinase and thrombomodulin was maintained. These modifications could help to improve graft survival by preventing thrombus formation on the surface of transplanted islets.

Noninvasive Imaging of Lipid Nanoparticle–Mediated Systemic Delivery of Small-Interfering RNA to the Liver

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.