Search

Improving drug delivery efficiency to solid tumor sites is a central challenge in anticancer therapeutic research. Our previous experimental study (Guo et al., Nat. Commun. 2018 , 9 , 130) showed that soft, elastic liposomes had increased uptake and accumulation in cancer cells and tumors in vitro and in vivo respectively, relative to rigid particles. As a first step toward understanding how liposomes' molecular structure and composition modulates their elasticity, we performed all-atom and coarse-grained classical molecular dynamics (MD) simulations of lipid bilayers formed by mixing a long-tailed unsaturated phospholipid with a short-tailed saturated lipid with the same headgroup. The former types of phospholipids considered were 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoleoyl- sn -glycero-3-phosphocholine (termed here DPMPC). The shorter saturated lipids examined were 1,2-diheptanoyl- sn -glycero-3-phosphocholine (DHPC), 1,2-didecanoyl- sn -glycero-3-phosphocholine (DDPC), 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC), and 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC). Several lipid concentrations and surface tensions were considered. Our results show that DOPC or DPMPC systems having 25-35 mol % of the shortest lipids DHPC or DDPC are the least rigid, having area compressibility moduli K A that are ∼10% smaller than the values observed in pure DOPC or DPMPC bilayers. These results agree with experimental measurements of the stretching modulus and lysis tension in liposomes with the same compositions. These mixed systems also have lower areas per lipid and form more uneven x - y interfaces with water, the tails of both primary and secondary lipids are more disordered, and the terminal methyl groups in the tails of the long lipid DOPC or DPMPC wriggle more in the vertical direction, compared to pure DOPC or DPMPC bilayers or their mixtures with the longer saturated lipid DLPC or DMPC. These observations confirm our hypothesis that adding increasing concentrations of the short unsaturated lipid DHPC or DDPC to DOPC or DPMPC bilayers alters lipid packing and thus makes the resulting liposomes more elastic and less rigid. No formation of lipid nanodomains was noted in our simulations, and no clear trends were observed in the lateral diffusivities of the lipids as the concentration, type of secondary lipid, and surface tension were varied.

Lysophosphatidic acid receptor 1 (LPAR1) is elevated in breast cancer. The deregulation of LPAR1, including the function and level of expression, is linked to cancer initiation, progression, and metastasis. LPAR1 antagonists, AM095 or Ki16425, may be effective therapeutic molecules, yet their limited water solubility hinders in vivo delivery. In this study, we report on the synthesis of two liposomal formulations incorporating AM095 or Ki16425, embedded within the lipid bilayer, as targeted nanocarriers for metastatic breast cancer (MBC). The data show that the Ki16425 liposomal formulation exhibited a 50% increase in internalization by MBC mouse epithelial cells (4T1) and a 100% increase in tumor accumulation in a mouse model of MBC compared with that of a blank liposomal formulation (control). At the same time, normal mouse epithelial cells (EpH-4Ev) internalized the Ki16425 liposomal formulation 25% lesser than the control formulation. Molecular dynamics simulations show that the integration of AM095 or Ki16425 modified the physical and mechanical properties of the lipid bilayer, making it more flexible in these liposomal formulations compared with liposomes without drug. The incorporation of an LPAR1 antagonist within a liposomal drug delivery system represents a viable therapeutic approach for targeting the LPA-LPAR1 axis, which may hinder the progression of MBC.

Liposomal drug delivery represents a highly adaptable therapeutic platform for treating a wide range of diseases. Natural and synthetic lipids, as well as surfactants, are commonly utilized in the synthesis of liposomal drug delivery vehicles. The molecular diversity in the composition of liposomes enables drug delivery with unique physiological functions, such as pH response, prolonged blood circulation, and reduced systemic toxicity. Herein, we discuss the impact of composition on liposome synthesis, function, and clinical utility., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Triple-negative breast cancer (TNBC), which has the highest mortality rate of all breast cancer, is in urgent need of a therapeutic that hinders the spread and growth of cancer cells. CRISPR genome editing holds the promise of a potential cure for many genetic diseases, including TNBC; however, its clinical translation is being challenged by the lack of safe and effective nonviral delivery systems for in vivo therapeutic genome editing. Here we report the synthesis and application of a noncationic, deformable, and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. We have demonstrated that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (Lcn2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. In an orthotopic TNBC model, we have shown that systemically administered tNLGs mediated >81% CRISPR knockout of Lcn2 in TNBC tumor tissues, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide experimental evidence that tNLGs can be used as a safe, precise, and effective delivery approach for in vivo CRISPR genome editing in TNBC., Competing Interests: Conflict of interest statement: P.G., J.Y., D.T.A., and M.A.M. are coinventors of a patent application filed by Boston Children’s Hospital (US patent application no. 62/472,104; filed on 16 March 2017). J.H. is a consultant for Simcere Pharmaceutical Company. The other authors declare no other competing interests.

The use of hydrogels in load bearing applications is often limited by insufficient toughness. 2-Hydroxyethyl methacrylate (HEMA) based hydrogels are appealing for translational work, as they are affordable and the use of HEMA is FDA approved. Furthermore, HEMA is photopolymerizable, providing spatiotemporal control over mechanical properties. We evaluated the ability of vinyl methacrylate (VM), allyl methacrylate (AM), and 3-(Acryloyloxy)-2-hydroxypropyl methacrylate (AHPM) to tune hydrogel toughness and Young's modulus. The crosslinkers were selected due to their heterobifunctionality (vinyl and methacrylate) and similar size and structure to EGDMA, which was shown previously to increase toughness as compared to longer crosslinkers. Vinyl methacrylate incorporation into HEMA hydrogels gave rise to hydrogels with Young's moduli spanning ranges for ligament to cartilage, with a peak toughness of 519 ± 70 kJ/m3 under physiological conditions. We report toughness (work of extension) as a function of modulus and equilibrium water content for all formulations. The hydrogels exhibited 80%-100% cell viability, which suggests they could be used in tissue engineering applications., Competing Interests: The authors have declared that no competing interests exist.

The plasticity of cancer epigenetics makes them plausible candidates for therapeutic intervention. We took advantage of elevated expression of lysophosphatidic acid receptor 1 (LPAR 1 ) in triple negative breast cancer (TNBC) tissues to target decitabine (DAC) and panobinostat (PAN) to breast cancer cells. DAC and PAN were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to increased expression of tumor suppressor genes and decreased expression of oncogenes. Although DAC and PAN have therapeutic benefits, they are limited by chemical instability and systemic toxicity. Herein, we present LPAR 1 -targeted, lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results demonstrated that the cell uptake and in vivo biodistribution of LNEs was dependent on LPAR 1 expression in TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth of mesenchymal breast cancer cells by restoring CDH1/E-cadherin and suppressing forkhead box M1 (FOXM1) expression. Epithelial breast cancer cells that inherently express low FOXM1 and high CDH1 were unaffected by DAC/PAN-LNEs. Overall, we successfully designed LPAR 1 -targeted LNEs that selectively act on CDH1(low)/FOXM1(high) TNBC cell lines.

Distinguishing malignant cells from non-neoplastic ones is a major challenge in triple-negative breast cancer (TNBC) treatment. Here, we developed a complementary targeting strategy that uses precisely matched, multivalent ligand-receptor interactions to recognize and target TNBC tumors at the primary site and metastatic lesions. We screened a panel of cancer cell surface markers and identified intercellular adhesion molecule-1 (ICAM1) and epithelial growth factor receptor (EGFR) as optimal candidates for TNBC complementary targeting. We engineered a dual complementary liposome (DCL) that precisely complements the molecular ratio and organization of ICAM1 and EGFR specific to TNBC cell surfaces. Our in vitro mechanistic studies demonstrated that DCLs, compared to single-targeting liposomes, exhibited increased binding, enhanced internalization, and decreased receptor signaling. DCLs consistently exhibited substantially increased tumor targeting activity and antitumor efficacy in orthotopic and lung metastasis models, indicating that DCLs are a platform technology for the design of personalized nanomedicines for TNBC.

Receptor-mediated drug delivery presents an opportunity to enhance therapeutic efficiency by accumulating drug within the tissue of interest and reducing undesired, off-target effects. In cancer, receptor overexpression is a platform for binding and inhibiting pathways that shape biodistribution, toxicity, cell binding and uptake, and therapeutic function. This review will identify tumor-targeted drug delivery vehicles and receptors that show promise for clinical translation based on quantitative in vitro and in vivo data. The authors describe the rationale to engineer a targeted drug delivery vehicle based on the ligand, chemical conjugation method, and type of drug delivery vehicle. Recent advances in multivalent targeting and ligand organization on tumor accumulation are discussed. Revolutionizing receptor-mediated drug delivery may be leveraged in the therapeutic delivery of chemotherapy, gene editing tools, and epigenetic drugs., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Atomic force microscopy (AFM) is a powerful tool to detect in vitro antibody-antigen interactions. To date, however, AFM-measured antibody-antigen interactions have yet to be exploited to predict in vivo tumor specificity of antibody-directed nanomedicines. In this study, we have utilized AFM to directly measure the biomechanical interaction between live triple negative breast cancer (TNBC) cells and an antibody against ICAM1, a recently identified TNBC target. For the first time, we provide proof-of-principle evidence that in vitro TNBC cell-ICAM1 antibody binding force measured by AFM on live cells more precisely correlates with in vivo tumor accumulation and therapeutic efficacy of ICAM1 antibody-directed liposomes than ICAM1 gene and surface protein overexpression levels. These studies demonstrate that live cell-antibody binding force measurements may be used as a novel in vitro metric for predicting the in vivo tumor recognition of antibody-directed nanomedicines.

Trachea replacement for nonoperable defects remains an unsolved problem due to complications with stenosis and mechanical insufficiency. While native trachea has anisotropic mechanical properties, the vast majority of engineered constructs focus on uniform cartilaginous-like conduits. These conduits often lack quantitative mechanical analysis at the construct level, which limits analysis of functional outcomes in vivo, as well as comparisons across studies. This review aims to present a clear picture of native tracheal mechanics at the tissue and organ level, as well as loading conditions to establish design criteria for trachea replacements. We further explore the implications of failing to match native properties with regards to implant collapse, stenosis, and infection.

To date, the role of elasticity in drug delivery remains elusive due to the inability to measure microscale mechanics and alter rheology without affecting chemistry. Herein, we describe the in vitro cellular uptake and in vivo tumor uptake of nanolipogels (NLGs). NLGs are composed of identical lipid bilayers encapsulating an alginate core, with tunable elasticity. The elasticity of NLGs was evaluated by atomic force microscopy, which demonstrated that they exhibit Young's moduli ranging from 45 ± 9 to 19,000 ± 5 kPa. Neoplastic and non-neoplastic cells exhibited significantly greater uptake of soft NLGs (Young's modulus <1.6 MPa) relative to their elastic counterparts (Young's modulus >13.8 MPa). In an orthotopic breast tumor model, soft NLGs accumulated significantly more in tumors, whereas elastic NLGs preferentially accumulated in the liver. Our findings demonstrate that particle elasticity directs tumor accumulation, suggesting that it may be a design parameter to enhance tumor delivery efficiency.

There remains no routine treatment for congenital tracheal abnormalities affecting more than 1/3 of the length. Natural and artificial prostheses are plagued by mechanical failure and inconsistent outcomes. Mimicking native tissue mechanics in an engineered replacement may improve functional and patient outcomes. We synthesized tubular constructs comprising photo-cross-linked methyl acrylate-co-methyl methacrylate, p(MA- co -MMA), with patterned r - and z -axes in order to achieve mechanical properties similar to lamb tracheae. Hard and soft alternating bands, and a soft vertical section, mimic tracheal architecture. Patterned constructs were capable of 46% elastic longitudinal extension. The construct longitudinal composite modulus, 0.34 ± 0.09 MPa, was not significantly different from ovine tracheae. The superior of two geometries evaluated supports up to a 46% reduction of internal volume within the physiological range of transmural pressures. Thus, these patterned hydrogels yielded longitudinal elasticity and radial rigidity while allowing for radial deformation required for effective coughing.

Identifying a molecular target is essential for tumor-targeted nanomedicine. Current cancer nanomedicines commonly suffer from poor tumor specificity, "off-target" toxicity, and limited clinical efficacy. Here, we report a method to screen and identify new molecular targets for tumor-targeted nanomedicine based on a quantitative analysis. In our proof-of-principle study, we used comparative flow cytometric screening to identify ICAM-1 as a potential target for metastatic melanoma (MM). We further evaluated ICAM-1 as a MM targeting moiety by characterizing its (1) tumor specificity, (2) expression level, (3) cellular internalization, (4) therapeutic function, and (5) potential clinical impact. Quantitation of ICAM-1 protein expression on cells and validation by immunohistochemistry on human tissue specimens justified the synthesis of antibody-functionalized drug delivery vehicles, which were benchmarked against appropriate controls. We engineered ICAM-1 antibody conjugated, doxorubicin encapsulating immunoliposomes (ICAM-Dox-LPs) to selectively recognize and deliver doxorubicin to MM cells and simultaneously neutralize ICAM-1 signaling via an antibody blockade, demonstrating significant and simultaneous inhibitory effects on MM cell proliferation and migration. This paper describes a novel, quantitative metric system that identifies and evaluates new cancer targets for tumor-targeting nanomedicine., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Breast cancer is the second leading cause of cancer-related mortality in women. Successful development of sensitive nanoprobes for breast cancer cell detection is of great importance for breast cancer diagnosis and symptomatic treatment. Herein, inspired by the intrinsic peroxidase property of gold nanoclusters, high loading, and targeting ability of ErbB2/Her2 antibody functionalized liposomes, we report that gold nanoclusters-loaded, target-directed, functionalized liposomes can serve as a robust sensing platform for amplified colorimetric detection of HER2-positive breast cancer cells. This approach allows HER2-positive breast cancer cell identification at high sensitivity with high selectivity. In addition, the colorimetric "readout" offers extra advantages in terms of low-cost, portability, and easy-to-use applications. The practicality of this platform was further proved by successful detection of HER2-positive breast cancer cells in human serum samples and in breast cancer tissue, which indicated our proposed method has potential for application in cancer theranostics., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Early detection of breast cancer is a critical component in patient prognosis and establishing effective therapy regimens. Here, we developed an easily accessible yet potentially powerful sensor to detect cancer cell targets by utilizing seven dual-ligand cofunctionalized gold nanoclusters (AuNCs) as both effective cell recognition elements and signal transducers. On the basis of this AuNC multichannel sensor, we have successfully distinguished healthy, cancerous and metastatic human breast cells with excellent reproducibility and high sensitivity. Triple negative breast cancer cells (TNBCs), which exhibit low expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2, were identified. The high accuracy of the blind breast cell sample tests further validates the practical application of the sensor array. In addition, the versatility of the sensor array is further justified by identifying amongst distinct cell types, different cell concentrations and cell mixtures. Notably, the drug-resistant cancer cells can also be efficiently discriminated. Furthermore, the dual-ligand cofunctionalized AuNCs can efficiently differentiate different cells from the peripheral blood of tumor-free and tumor-bearing mice. Taken together, this fluorescent AuNCs based array provides a powerful cell analysis tool with potential applications in biomedical diagnostics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The linker between the targeting moiety and the nanoparticle is often overlooked when engineering targeted drug delivery vehicles. We hypothesized that pH-triggered conformational changes of an elastin-like peptide (ELP) linker, with repeating VPGVG sequences, could alter the binding affinity of the well-established targeting moiety arginine-glycine-aspartic acid (RGD), which is known to enhance the delivery of nanoparticles to tumor cells via integrin overexpression. The pH change from blood (pH 7.4) to the tumor environment (pH 6) was used to elicit a conformational change in the ELP linker, as described by circular dichroism. Atomic force microscopy confirmed that RGD-ELP resulted in stronger adhesion to both MDA-MB-231 and HCC1806 breast cancer cells at pH 6 relative to pH 7.4. No change in adhesion force was measured as a function of pH for the non-neoplastic MCF-10A cell line and the nontargeting GDR-ELP peptide. This translated to significant binding and uptake of RGD-ELP modified liposomes at pH 6.0 relative to pH 7.4. These results indicate that the pH-triggered conformational structure of the ELP linker shifts RGD-mediated cancer cell targeting from non-active (pH 7.4) to active (pH 6). The reversible shift in ELP secondary structure may be used to engineer targeted drug delivery vehicles with tunable uptake.

Early and accurate diagnosis of breast cancer holds great promise to improve treatability and curability. Here, we report the usage of six luminescent nanodot-graphene oxide complexes as novel fluorescent nanoprobes in a sensing array capable of effectively identifying healthy, cancerous, and metastatic human breast cells. The sensory system is based on the utilization of nanoprobe-graphene oxide sensor elements that can be disrupted in the presence of breast cells to give fluorescent readouts. Using this multichannel sensor, we have successfully identified breast cancer cells and distinguished between estrogen receptor positive, human epidermal growth factor receptor-2 positive, and triple negative phenotypes. This approach also allows cell identification at high sensitivity (200 cells) with high reproducibility. The unknown cell sample analysis indicates that the sensor is able to identify 49 out of 50 breast cell samples correctly, with a detection accuracy of 98%. Taken together, this array-based luminescent nanoprobe-graphene oxide sensing platform presents a useful cell screening tool with potential applications in biomedical diagnostics., (Copyright © 2016. Published by Elsevier B.V.)

Leukocyte recruitment plays a key role in chronic inflammatory diseases such as cardiovascular disease, rheumatoid arthritis, and cancer. Leukocyte rolling and arrest are mediated in part by the temporally-regulated surface expression of vascular cell adhesion molecule-1 (VCAM1) on endothelial cells (ECs). In this paper, we engineered a pH-responsive vehicle comprised of 30 mol% dimethylaminoethyl methacrylate (30D) and 70 mol% hydroxyethyl methacrylate (70H) to encapsulate, protect, and deliver VCAM1 small interfering RNA (siRNA). The ability of siRNA to reduce VCAM1 gene expression is in direct opposition to its activation by cytokines. At 12 h post-activation, VCAM1 gene knockdown was 90.1 ± 7.5% when delivered via 30D/70H nanoparticles, which was on par with a leading commercial transfection agent. This translated into a 68.8 ± 6.7% reduction in the surface density of VCAM1 on cytokine-activated ECs. The pH-responsive delivery of VCAM1 siRNA efficiently reduced temporal surface protein expression, which may be used to avert leukocyte recruitment.

Tracheal damage, abnormality or absence can result from the growth of tumors or from Congenital High Airway Obstruction Syndrome. No optimal or routine treatment has been established for tracheal repair, despite numerous attempts with natural and artificial prostheses. The fetal trachea is comprised of cartilaginous rings connected by an elastomeric tissue. In an effort to design an engineered trachea replacement, we have synthesized 2-hydroxyethyl methacrylate hydrogels with moduli of 67 ± 3.1 kPa (soft) and 13.0 ± 1.8 MPa (hard). Given the criteria for longitudinal extensibility and lateral rigidity applied during respiration, we evaluated a series of patterned hydrogels with different sizes of hard and soft segments to mimic fetal tracheas. A 1:2 ratio of soft:hard segments resulted in a construct capable of 11.0 ± 1% extension within the elastic range. Tubular constructs with this ratio required similar load/length for cyclic compression as ovine trachea samples. Achieving biomimetic mechanical properties in a trachea replacement may be essential for achieving normal respiration in recipient patients., Statement of Significance: Fetal abnormalities or tumors can result in tracheal absence or damage. Despite numerous attempts with natural and artificial replacements, there is still no routine treatment for tracheal repair. The literature recognizes the importance of tracheal lateral rigidity and longitudinal extensibility for normal respiration. Achieving closely matched mechanical properties may provide proper function and help decrease implant fibrosis and subsequent occlusion. In this study, we evaluated the mechanics of a series of patterned, tubular hydrogels with different ratios of hard and soft segments to mimic alternating cartilage and ligament sections in fetal tracheas. We compared our results to that of sheep trachea. This is the first report to assess both radial rigidity and longitudinal extensibility in an engineered trachea construct., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Lipocalin 2 (Lcn2) is a promising therapeutic target as well as a potential diagnostic biomarker for breast cancer. It has been previously shown to promote breast cancer progression by inducing the epithelial to mesenchymal transition in breast cancer cells as well as by enhancing angiogenesis. Lcn2 levels in urine and tissue samples of breast cancer patients has also been correlated with breast cancer status and poor patient prognosis. In this study, we have engineered a novel liposomal small interfering RNA (siRNA) delivery system to target triple negative breast cancer (TNBC) via a recently identified molecular target, intercellular adhesion molecule-1 (ICAM-1). This ICAM-1-targeted, Lcn2 siRNA- encapsulating liposome (ICAM-Lcn2-LP) binds human TNBC MDA-MB-231cells significantly stronger than non-neoplastic MCF-10A cells. Efficient Lcn2 knockdown by ICAM-Lcn2-LPs led to a significant reduction in the production of vascular endothelial growth factor (VEGF) from MDA-MB-231 cells, which, in turn, led to reduced angiogenesis both in vitro and in vivo. Angiogenesis (neovascularization) is a requirement for solid tumor growth and progression, and its inhibition is an important therapeutic strategy for human cancers. Our results indicate that a tumor-specific strategy such as the TNBC-targeted, anti-angiogenic therapeutic approach developed here, may be clinically useful in inhibiting TNBC progression.

The pitfall of all chemotherapeutics lies in drug resistance and the severe side effects experienced by patients. One way to reduce the off-target effects of chemotherapy on healthy tissues is to alter the biodistribution of drug. This can be achieved in two ways: Passive targeting utilizes shape, size, and surface chemistry to increase particle circulation and tumor accumulation. Active targeting employs either chemical moieties (e.g. peptides, sugars, aptamers, antibodies) to selectively bind to cell membranes or responsive elements (e.g. ultrasound, magnetism, light) to deliver its cargo within a local region. This article will focus on the systemic administration of anti-cancer agents and their ability to home to tumors and, if relevant, distant metastatic sites., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The CXCR4 receptor triggers cell migration and, in breast cancer, promotes metastasis. To date, the dynamic assembly of CXCR4 on the cell surface as a mediator of receptor binding is not well characterized. The objective of this work is to quantify the density, spatial organization, and magnitude of binding of the CXCR4 receptor on live metastatic breast cancer (MBC) cells. We measured the Young's modulus, the CXCR4 surface density, and CXCR4 unbinding force on MBC cells by atomic force microscopy. We conclude that the CXCR4 density, spatial organization, and matrix stiffness are paramount to achieve strong binding., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A principal challenge in wound healing is a lack of cell recruitment, cell infiltration, and vascularization, which occurs in the absence of temporal and spatial cues. We hypothesized that a scaffold that expands due to local changes in pH may alter oxygen and nutrient transport and the local cell density, leading to enhanced cell deposition and survival. In this study, we present a pH-responsive scaffold that increases oxygen transport, as confirmed by our finite element model analysis, and cell proliferation relative to a non-responsive scaffold. In vivo, responsive scaffolds induce a pro-healing gene expression profile indicative of enhanced angiogenesis, granulation tissue formation, and tissue remodeling. Scaffolds that stretch in response to their environment may be a hallmark for tissue regeneration., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Liposomes may be engineered to target inflamed endothelium by mimicking ligand-receptor interactions between leukocytes and cytokine-activated endothelial cells (ECs). The upregulation and assembly of vascular cell adhesion molecule-1 (VCAM1) and E-selectin on the cell membrane upon exposure to cytokines have shown potential for drug delivery vehicles to target sites of chronic endothelial inflammation, such as atherosclerosis and cancer. Herein, we characterized EC surfaces by measuring the E-selectin and VCAM1 surface densities and adhesion forces of aVCAM1 and aE-selectin to ECs. We quantified the antibody density, ratio, and diffusivity of liposomes to achieve significant binding and internalization. At 1 h, the 1:1 ratio of VCAM1:E-selectin antibodies was significantly higher than 1:0 and 0:1. Significant binding and uptake was achieved at aE-selectin densities as low as 400 molecules/μm(2). The highest levels of binding and uptake were achieved when using a 1:1 ratio of VCAM1:E-selectin antibodies at a density of 1000 molecules/μm(2); this density is 85% lower than previous reports. The binding and uptake of functionalized liposomes were reduced to levels comparable to IgG functionalized liposomes upon a 10-fold reduction in liposome membrane diffusivity. We conclude with a liposomal design that discriminates between healthy and inflamed endothelium while reducing antibody surface presentation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Triple negative breast cancers (TNBCs) have a high mortality rate owing to aggressive proliferation and metastasis and a lack of effective therapeutic options. Herein, we describe the overexpression of intercellular adhesion molecule-1 (ICAM-1) in human TNBC cell lines and tissues, and demonstrate that ICAM-1 is a potential molecular target and biomarker for TNBC therapy and diagnosis. We synthesized ICAM-1 antibody-conjugated iron oxide nanoparticles (ICAM-IONPs) as a magnetic resonance imaging (MRI) probe to evaluate tumor targeting. Quantitative analysis of ICAM-1 surface expression predicted the targeting capability of ICAM-IONPs to TNBC cells. MRI of the TNBC xenograft tumor after systemic administration of ICAM-IONPs, coupled with iron quantification and histology, demonstrated a significant and sustained MRI contrast enhancement and probe accumulation in tumors with ICAM-1 overexpression relative to control. Identification of ICAM-1 as a TNBC target and biomarker may lead to the development of a new strategy and platform for addressing a critical gap in TNBC patient care.

Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach--coupling the CXCR4 axis blockade with Lcn2 silencing--significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC.

The primary cause of mortality in breast cancer is tumor aggressiveness, characterized by metastases to regional lymph nodes, bone marrow, lung, and liver. C-X-C chemokine receptor type 4 (CXCR4) has been shown to mobilize breast cancer cells along chemokine gradients. Quantification of CXCR4 surface expression may predict the efficacy of anti-CXCR4 labeled liposomal therapeutics to target and kill breast cancer cells. We evaluated gene and surface receptor expression of CXCR4 on breast cancer cell lines distinguished as having low and high invasiveness, MDA-MB-175VII and HCC1500, respectively. CXCR4 surface expression did not correlate with invasiveness. MDA-MB-175VII exhibited more binding to anti-CXCR4 labeled liposomes relative to HCC1500. Increased binding correlated with greater cell death relative to IgG labeled liposomes. Quantitative cell characterization may be used to select targeted therapeutics with enhanced efficacy and minimal side effects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Limited characterization of how the stem cell niche evolves has hindered our ability to mimic the physiological environment. In this paper, we hypothesized that hypoxia-induced extracellular matrix (ECM) cues may facilitate cardiomyogenesis. We evaluated the expression of four ECM proteins - fibronectin, collagen I, collagen IV, and laminin - over a period of 20 days in H1 and H9 human embryonic stem cell-derived embryoid bodies (EBs) under hypoxic (5% oxygen) and normoxic (21% oxygen) conditions. Hypoxic EBs exhibited increased collagen I, collagen IV and fibronectin expression relative to normoxic EBs between days 9-13, which coincided with increased expression of mesoderm genes. The effect of ECM cues was confirmed by plating day 9 EBs on collagen IV, gelatin, and fibronectin-rich substrates for 11 days. Hypoxia/gelatin cultures synergistically increased the cardiomyocyte yield by 1.7 and 5.5 fold relative to normoxia/gelatin and normoxia/collagen IV cultures, respectively. Current differentiation protocols may underestimate the contribution of hypoxia and ECM cues that evolve during EB maturation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Materials that adhere to the endothelial cell (EC) lining of blood vessels may be useful for treating vascular injury. Cell adhesion molecules (CAMs), such as endothelial leukocyte adhesion molecule-1 (E-selectin) and vascular cell adhesion molecule-1 (VCAM1), modulate EC-leukocyte interactions. In this study, we mimicked cell-cell interactions by seeding cells on alginate hydrogels modified with antibodies against E-selectin and VCAM1, which are upregulated during inflammation. ECs were activated with interleukin-1α to increase CAM expression and subsequently seeded onto hydrogels. The strength of cell adhesion onto gels was assessed via a centrifugation assay. Strong, cooperative EC adhesion was observed on hydrogels presenting a 1:1 ratio of anti-VCAM1:anti-E-selectin. Cell adhesion was stronger on dual functionalized gels than on gels modified with anti-VCAM1, anti-E-selectin or the arginine-glycine-aspartic acid (RGD) peptide alone. Anti-VCAM1:anti-E-selectin-modified hydrogels may be engineered to adhere the endothelium cooperatively., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell adhesion molecules govern leukocyte-endothelial cell (EC) interactions that are essential in regulating leukocyte recruitment, adhesion, and transmigration in areas of inflammation. In this paper, we synthesized hydrogel matrices modified with antibodies against vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte adhesion molecule-1 (E-Selectin) to mimic leukocyte-EC interactions. Adhesion of human umbilical vein ECs to polyvinyl alcohol (PVA) hydrogels was examined as a function of the relative antibody ratio (anti-VCAM1:anti-E-Selectin) and substrate elasticity. Variation of PVA backbone methacrylation was used to affect hydrogel matrix stiffness, ranging from 130 to 720 kPa. Greater EC adhesion was observed on hydrogels presenting 1:1 anti-VCAM1:anti-E-Selectin than on gels presenting either arginine-glycine-asparagine (RGD) peptide, anti-VCAM1, or anti-E-Selectin alone. Engineered cell adhesion - based on complementing the EC surface presentation - may be used to increase the strength of EC-matrix interactions. Hydrogels with tunable and synergistic adhesion may be useful in vascular remodeling., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Inflammation is in part defined by the transient upregulation of cell adhesion molecules on the surface of endothelial cells (ECs) in response to cytokines. We hypothesized that liposomes with a complementary surface presentation of antibodies to the pattern of molecules on the EC surface may enhance targeting. We quantified the expression of vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte cell adhesion molecule-1 (E-selectin) on ECs upon exposure to either tumor necrosis factor-α (TNF-α) or interleukin-1α (IL-1α) as a function of time. Liposomes, composed of 95 mol% dioleoyl phosphatidylcholine (DOPC) and 5 mol% dodecanyl phosphatidylethanolamine (N-dod-PE), were prepared by conjugating different molar ratios of antibodies against VCAM1 (aVCAM1) and E-selectin (aE-selectin). Increased binding was observed when immunoliposomes complemented the presentation of VCAM1:E-selectin expressed on TNF-α activated ECs. The 1:1 aVCAM1:aE-selectin liposomes had maximal binding at both 6 and 24 h on IL-1α activated ECs due to differences in molecular organization. The results demonstrate that liposomes targeting to inflamed endothelium may be optimized by exploiting the dynamic expression of VCAM1 and E-selectin on the EC surface., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inspired by morphogenesis in biology, we present a strategy for developing functional 3D materials with the capacity to morph based on environmental cues. We utilized local mechanical stresses to cause global shape changes in colloidosomes. Colloidosomes were assembled from pH-sensitive calcium alginate particles (CAPs) with high and low swelling ratios. Colloidosomes were subsequently cross-linked via diamine compounds with varying carbon chain lengths. New colloidosome isoforms were generated from heterogeneous mixtures of CAPs, which resulted in nonuniform stresses. Our study demonstrated that coordinated networks of heterogeneous subunits may be used to design programmable materials., (© 2011 American Chemical Society)

Scaffolds that couple electrical and elastic properties may be valuable for cardiac cell function. However, existing conductive materials do not mimic physiological properties. We prepared and characterized a tunable, hybrid hydrogel scaffold based on Au nanoparticles homogeneously synthesized throughout a polymer templated gel. Conductive gels had Young's moduli more similar to myocardium relative to polyaniline and polypyrrole, by 1-4 orders of magnitude. Neonatal rat cardiomyocytes exhibited increased expression of connexin 43 on hybrid scaffolds relative to HEMA with or without electrical stimulation.

For years, the field of drug delivery has focused on (1) controlling the release of a therapeutic and (2) targeting the therapeutic to a specific cell type. These research endeavors have concentrated mainly on the development of new degradable polymers and molecule-labeled drug delivery vehicles. Recent interest in biomaterials that respond to their environment have opened new methods to trigger the release of drugs and localize the therapeutic within a particular site. These novel biomaterials, usually termed "smart" or "intelligent", are able to deliver a therapeutic agent based on either environmental cues or a remote stimulus. Stimuli-responsive materials could potentially elicit a therapeutically effective dose without adverse side effects. Polymers responding to different stimuli, such as pH, light, temperature, ultrasound, magnetism, or biomolecules have been investigated as potential drug delivery vehicles. This review describes the most recent advances in "smart" drug delivery systems that respond to one or multiple stimuli.

Lipid rafts are plasma membrane microdomains rich in cholesterol, sphingolipids, and cell surface receptors. Recent studies demonstrated the upregulation and localization of two receptors, intercellular cell adhesion molecule-1 (ICAM, CD54) and endothelial leukocyte adhesion molecule-1 (E-selectin, CD64E), within lipid raft microdomains of inflamed or injured endothelial cells (ECs). We hypothesized that the localization of ICAM and E-selectin within lipid rafts may be essential for drug delivery vehicles labeled with antibodies against ICAM (aICAM) and E-selectin (aE-selectin). To eliminate localization of cell surface receptors, ECs were treated with a cholesterol depleting drug, methyl-β-cyclodextrin. We also tested if antibody mobility and the ratio of aICAM to aE-selectin on immunoliposomes influenced binding to lipid-raft-depleted cells. Liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m) = -20 °C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m) = 42 °C) which are in the liquid crystalline and gel phase at 37 °C, respectively. Mobility and the aICAM:aE-selectin ratio influenced cellular binding only when lipid rafts form. In the absence of lipid rafts, cellular binding of both DOPC and DPPC immunoliposomes was reduced to the nonspecific binding level. These results, which were obtained under static conditions, suggest that the presence of lipid rafts in ECs is critical for targeted drug delivery.

We synthesized a series of pH-sensitive vehicles, composed of dimethylaminoethyl methacrylate (DMAEMA) and 2-hydroxyethyl methacrylate (HEMA), to optimize the triggered release of DNA for gene transfection. The purpose of this study was to assess the role of swelling and cationic character independently on transfection; both of which may affect DNA release. Gene transfection was performed by delivering plasmid DNA (pDNA) encoding for luciferase. DNA release was controlled via volumetric swelling by regulating the endosomal pH as a result of inhibiting V ATPases using bafilomycin A1. Increasing the cationic character from 10 to 30 mol% DMAEMA did not increase transfection when swelling was inhibited. Transfection was significantly affected by the rate of pDNA release. pH-sensitive nanocarriers were also compared to vehicles comprised of polyethyleneimine (PEI), dioleoyl triammonium propane (DOTAP), and poly(lactic-co-glycolic acid) (PLGA, 50:50). pDNA encapsulating DMAEMA/HEMA nanoparticles and PEI/pDNA complexes had reduced transfection when V ATPases were inhibited, whereas pDNA encapsulating PLGA nanoparticles showed no endosomal pH dependence. DMAEMA/HEMA nanoparticles cross-linked with 3 mol% tetraethylene glycol dimethacrylate (TEGDMA) reported equivalent or greater gene transfection relative to the nanocarriers tested at 24 and 48 h., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Coupling molecular sensing with electrical conductivity may provide an important and valuable resource in assessing disease pathology. Here, we introduce pH-responsive hydrogels with homogeneously synthesized gold (Au) nanoparticles that reversibly alter conductivity through pH-induced volumetric swelling. These intelligent hybrid materials respond to physiological pH shifts (pH 7.4 to 5.5) that can (1) alter the conductivity of the gel or (2) create conductive conduits via micropatterned arrays.

Targeted drug delivery to inflamed or injured vascular endothelial cells (ECs) and smooth muscle cells (SMCs) may provide a precise and effective therapeutic treatment for cardiovascular diseases. Upregulation of cytokine-regulated cell surface receptors, intercellular cell adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM), on ECs and SMCs are used to target drug delivery vehicles. Recent studies demonstrate clustering of these molecules in lipid rafts may affect binding due to a nonhomogenous presentation of antibodies. We hypothesized that altering the antibody ratio for ICAM and ELAM (aICAM:aELAM) and mobility would influence cellular targeting. To alter antibody mobility, liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m)=-20 degrees C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m)=42 degrees C) which are in the liquid crystalline (L(alpha)) and gel phase (L(beta)) at 37 degrees C, respectively. We report that cellular binding of DOPC immunoliposomes by ECs is maximal at an equimolar ratio of aICAM:aELAM whereas DPPC immunoliposomes showed no ratio dependence and binding was reduced by more than 2-fold. SMCs, which do not express ELAM, show a dependence on aICAM surface density. These results suggest that antibody mobility and molar ratio play a key role in increasing receptor-mediated cell targeting.

Treatment of diseases on the molecular level by genetic material is limited by effective delivery mechanisms. We focused on the synthesis of a pH-sensitive gene delivery vehicle based on dimethylaminoethyl methacrylate (DMAEMA) with tunable swelling, cross-linking density, and DNA release kinetics within the endosomal pH range. Our strategy, which utilized a single step for DNA encapsulation, enhanced gene transfection efficiency and reduced cytotoxicity relative to polyethyleneimine (PEI) and poly-L-lysine (PLL).

Fluidic devices are often made by irreversibly bonding a polydimethylsiloxane (PDMS) mold to itself or a glass substrate by plasma treatment. This method limits the range of materials for fluidic device fabrication and utility for subsequent processing. Here, we present a simple and inexpensive method to fabricate fluidic devices using magnets to reversibly adhere PDMS and other polymer matrices to glass or gel substrates. This approach enables fluidic devices to be fabricated from a variety of materials other than PDMS and glass. Moreover, this method can be used to fabricate composite devices, three-dimensional scaffolds and hydrogel-based fluidic devices.

Embryonic stem cells and induced pluripotent stem cells have the potential to be a renewable source of cardiomyocytes for use in myocardial cell replacement strategies. Although progress has been made towards differentiating stem cells to specific cell lineages, the efficiency is often poor and the number of cells generated is not suitable for therapeutic usage. Recent studies demonstrated that controlling the stem cell microenvironment can influence differentiation. Components of the extracellular matrix are important physiological regulators and can provide mechanical cues, direct differentiation and improve cell engraftment into damaged tissue. Bioreactors are used to control the microenvironment and produce large numbers of desired cells. This article describes recent methods to achieve cardiomyocyte differentiation by engineering the stem cell microenvironment. Successful translation of stem cell research to therapeutic applications will need to address large-scale cardiomyocyte differentiation and purification, assessment of cardiac function and synchronization, and safety concerns.

Differentiation of human embryonic stem (hES) cells into cells for regenerative medicine is often initiated by embryoid body (EB) formation. EBs may be treated with soluble biochemicals such as cytokines, growth factors and vitamins to induce differentiation. A scanning electron microscopy analysis, conducted over 14 days, revealed time-dependent changes in EB structure which led to the formation of a shell that significantly reduced the diffusive transport of a model molecule (374 Da) by >80%. We found that the shell consists of 1) an extracellular matrix (ECM) comprised of collagen type I; 2) a squamous cellular layer with tight cell-cell adhesions associated with E-cadherin; and 3) a collagen type IV lining indicative of a basement membrane. Disruption of the basement membrane, by either inhibiting its formation with noggin or permeabilizing it with collagenase, resulted in recovery of diffusive transport. Increasing the diffusive transport of retinoic acid (RA) and serum in EBs by a 15-min collagenase digestion on days 4, 5, 6 and 7 promoted neuronal differentiation. Flow cytometry and quantitative RT-PCR analysis of collagenase-treated EBs revealed 68% of cells expressing neural cell adhesion molecule (NCAM) relative to 28% for untreated EBs. Our results suggest that limitations in diffusive transport of biochemicals need to be considered when formulating EB differentiation strategies.

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16+/-0.2 and 0.32+/-0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06+/-0.02 and 0.14+/-0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.

Surface rheology of irreversibly bound hydrophobically modified poly(ethylene glycol) (PEG) polymers (HMPEG) on a dipalmitoylphosphatidylcholine (DPPC) monolayer is investigated to determine attributes that may contribute to immune recognition. Previously, three comb-graft polymers (HMPEG136-DP3, HMPEG273-DP2.5, and HMPEG273-DP5) adsorbed on liposomes were examined for their strength of adsorption and protection from complement binding. The data supported an optimal ratio between the hydrophilicity of the PEG polymer and the number of hydrophobic anchors. The HMPEG polymers have different polymer brush thicknesses (4.2-5.9 nm) and levels of cooperativity (2.5-5 hydrophobes). The results indicate that an increased viscous force (above 0.25 mN s/m) at the surface may enable the polymers to shield liposomes from protein interactions. Similar rheological behavior is shown for all polymer architectures at low polymer surface coverage (0.5 mg/m2, in the mushroom regime), whereas at high surface coverage (>0.5 mg/m2, in the brush regime), we observe a structural dependence of the surface viscous forces at 40 mN/m. This threshold correlates with a 92% decrease in complement protein binding for liposomes coated with 1 mg/m2 HMPEG273-DP5. This may suggest that surface viscous forces play a role in reducing complement protein binding.

pH-Sensitive poly(N,N-dimethylaminoethyl methacrylate (DMAEMA)/2-hydroxyethyl methacrylate (HEMA)) nanoparticles were prepared for the triggered release of paclitaxel within a tumor microenvironment. Tumors exhibit a lower extracellular pH than normal tissues. We show that paclitaxel release from DMAEMA/HEMA particles can be actively triggered by small, physiological changes in pH (within 0.2-0.6 pH units). Monodispersed nanoparticles were synthesized by forming an O/W emulsion followed by photopolymerization. Particles were characterized by transmission electron microscopy, dynamic light scattering, electrophoresis, and cytotoxicity. High release rates and swelling ratios are achieved at low pH, low crosslinking density, and high content of DMAEMA. Paclitaxel release is limited to 9% of the payload at pH 7.4 after a 2-h incubation at 37 degrees C. After adjusting to pH 6.8, 25% of the payload is released within 2h. Cell viability studies indicate that pH-sensitive DMAEMA/HEMA nanoparticles are not cytotoxic and may be used as an efficient, feedback-regulated drug delivery carrier.

Triggered release of adsorbed polymers from liposomes enables protection against immune recognition during circulation and subsequent intracellular delivery of DNA. Polycationic blocks, poly[2-(dimethylamino) ethyl methacrylate] (DMAEMA) (0.8, 3.1, 4.9, or 9.8 kg/mol) or polylysine (K) (3 kg/mol), act as anchors for poly(ethylene glycol) (PEG) (2 or 5 kg/mol) protective blocks. In addition, a copolymer with 15 strictly alternating blocks of PEG (2 kg/mol) and cationic amine sites was evaluated as a protective coating. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane, a titratable lipid with a pKa of approximately 6.7, allows the liposome's net charge to increase as the pH shifts from 7.4 in the bloodstream to 5.5 in the endosome. The increased net liposome cationicity results in decreased cationic polymer adsorption. The EMPEG113-DMAEMA31 and EMPEG113-DMAEMA62 association constants decrease from 3.1 and 6.2 (mg/m(2))/(mg/ml) at pH 7.4 to 1.7 and 3.2 (mg/m(2))/(mg/ml) at pH 5.5, respectively. However, EMPEG45-DMAEMA5, EMPEG45-DMAEMA20, and EMPEG45-N-DP15 did not show a strong response to changes in pH. Cationic polymer adsorption exceeds calculated values for liposome neutralization, resulting in adsorption profiles in the brush regime.

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml). The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.