Soon-Mi Lim

Polymeric Nanostructures for Imaging and Therapy.

Medical diagnosis and therapy are essential for providing patients with proper care, although inefficient diagnosis and therapy are usually associated with either improper detection of the diseases, unsatisfactory therapeutic outcomes and/or serious adverse reactions. Advances in the design of various diagnostic and therapeutic agents, and the recent trend of utilizing molecules for both therapeutic and diagnostic applications (i.e. theranostics), still have not achieved the maximum benefits of controlling the navigation and biodistribution of these molecules within the biological system. The key challenges towards the use of these agents, from small molecules to macromolecular drugs (e.g. natural, proteins and nucleic acids-based drugs, or synthetic, polymer-based conjugates, carriers or other systems), are, for example, the loss of activity via rapid clearance or degradation, inefficient delivery to the target sites, and inappropriate probing of the disease states, dependent on the particular disease and its location in the body. The concept of nanotechnology has been initiated early in 1959 by Richard Feynman in his famous historical talk at Caltech “There’s Plenty of Room at the Bottom”, with introduction of the possibility of manipulating materials at the atomic and molecular levels.1 In 1974, Norio Taniguchi, at Tokyo University, first utilized the term “nanotechnology” referring to the design of materials on the nanoscale.2 In the early 1990’s and until now, the use of nanomaterials of different nature (organic and inorganic), and for various applications (multiple disciplines) has been greatly expanded, in particular, over the last couple of decades.3-4 In the medical field, nanotechnology has emerged to include non-invasive systems for probing of disease and also capable of carrying cargo for localized high concentration delivery, known as “nanomedicine”, with reduction of off-target effects. The use of nanomaterials, in particular polymeric nanostructures, has demonstrated efficiency in improving delivery of diagnostic and therapeutic agents to the target sites, and the feasibility of incorporating several therapeutic/diagnostic/targeting moieties within specific compartments of the nanoparticles, with control of their navigation in the body and to the target sites. Further understanding of the nature and microenvironments of biological systems (e.g. different pH, temperature, permeability, drainage, or overexpressing proteins, enzymes or receptors), and the barriers towards the delivery of various moieties to their destinations, which could be either intra- or extracellular, has aided the design of nanomaterials that could evade the various physiological barriers. Selective delivery to the site of the disease can increase the therapeutic efficacy, imaging contrast and accuracy, reduce adverse reactions, and reduce the dose and cost of medications. Initially, platform technologies were the target for nanostructure designs, but with the complications of biological systems, it has been recognized over the past decade that disease- and patient-specific medical treatment is needed for efficacy—this review highlights a few examples developed within the past couple of years, with a focus on in vivo studies together with novel designs and significant advances in syntheses. The advantages of polymeric nanostructures over other types of nanomaterials are based upon the flexibility over which their structures can be modified to yield materials of various compositions, morphologies, sizes, surface properties, with possibility of hierarchical assembly of several nanomaterials of various components into one construct that can be accommodated with a variety of therapeutic, diagnostic and/or targeting moieties, within selective compartments of the nanodevices. High efficiency in diagnosis and treatment of diseases and improving patient quality of life and compliance can be achieved through understanding the molecular events associated with various diseases, and combining the advances in the design of therapeutic and diagnostic agents and nanomaterials, together with the innovative instruments utilized for monitoring these agents. This review will focus on several recent advances in the design of polymeric nanoparticles that have been utilized for delivery of diagnostic and/or therapeutic agents, and the various barriers towards the clinical development of these materials. After a brief overview of the capabilities and challenges with medical imaging and therapy, in general, disease-specific examples of polymer nanoparticles designed specifically to overcome the challenges and address unmet medical needs will be discussed in detail.

Specific anion effects on water structure adjacent to protein monolayers.

Vibrational sum frequency spectroscopy (VSFS) was used to explore specific ion effects on interfacial water structure adjacent to a bovine serum albumin (BSA) monolayer adsorbed at the air/water interface. The subphase conditions were varied by the use of six different sodium salts and four different pH values. At pH 2 and 3, the protein layer was positively charged and it was found that the most chaotropic anions caused the greatest attenuation of water structure. The order of the salts followed an inverse Hofmeister series. On the other hand, when the protein layer was near its isoelectric point (pH 5), the most chaotropic anions caused the greatest increase in water structure, although the effect was weak. In this case, a direct Hofmeister series was obeyed. Finally, virtually no effect was observed when the protein layer was negatively charged with a subphase pH of 9. For comparison, similar experiments were run with positively charged, negatively charged, and zwitterionic surfactant monolayers. These experiments gave rise to nearly the identical results as the protein monolayer which suggested that specific anion effects are dominated by the charge state of the interfacial layer rather than its detailed chemical structure. In a final set of experiments, salt effects were examined with a monolayer made from an elastin-like polypeptide (ELP). The peptide consisted of 120 pentameric repeats of the sequence Val-Pro-Gly-Val-Gly. Data from this net neutral biopolymer followed a very weak, but direct Hofmeister series. This suggested that direct anion binding to the amide groups in the backbone of a polypeptide is quite weak in agreement with the BSA data. The results from the variously charged protein, surfactant, and polymer monolayers were compared with a modified Gouy-Chapman-Stern model. The agreement with this simple model was quite good.

Integrated microscopy for real-time imaging of mechanotransduction studies in live cells

Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells.

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5h, while a significant inhibition was recorded at 24h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.

Live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

Rapidly-cured isosorbide-based cross-linked polycarbonate elastomers

The rapid synthesis of an optically-transparent, flexible elastomer was performed utilizing the naturally-derived source, isosorbide. A novel monomer based on isosorbide (isosorbide dialloc, IDA) was prepared by installing carbonate functionalities along with external olefins for use in thiol–ene click chemistry. Cross-linked networks were created using the commercially-available cross-linker, trimethylolpropane tris(3-mercaptopropionate) (TMPTMP) and resulted in IDA-co-TMPTMP, an optically-transparent elastomer. Systematically, IDA-co-TMPTMP networks were synthesized using a photoinitiator, a UV cure time of one minute and varied post cure times (0–24 h, 125 mm Hg) at 100 °C to observe effects on mechanical, thermal and surface alterations. The mechanical properties also had limited changes with post cure time, including a modulus at 25 °C of 1.9–2.8 MPa and an elongation of 220–344%. The thermal decomposition temperatures of the networks were consistent, ca. 320 °C, while the glass transition temperature remained below room temperature for all samples. A cell viability assay and fluorescence imaging with adherent cells are also reported in this study to show the potential of the material as a biomedical substrate. A degradation study for 60 days resulted in 8.3 ± 3.5% and 97.7 ± 0.3% mass remaining under accelerated (1 M NaOH, 60 °C) and biological conditions (pH 7.4 PBS at 37 °C), respectively. This quickly-synthesized material has the potential to hydrolytically degrade into biologically-benign and environmentally-friendly by-products and may be utilized in renewable plastics and/or bioelastomer applications.

Harnessing the Chemical Diversity of the Natural Product Magnolol for the Synthesis of Renewable, Degradable Neolignan Thermosets with Tunable Thermomechanical Characteristics and Antioxidant Activity

Magnolol, a neolignan natural product with antioxidant properties, contains inherent, orthogonal, phenolic, and alkenyl reactive groups that were used in both direct thermoset synthesis, as well as the stepwise synthesis of a small library of monomers, followed by transformation into thermoset materials. Each monomer from the small library was prepared via a single step functionalization reaction of the phenolic groups of magnolol. Thermoset materials were realized through solvent-free, thiol-ene reactions, and the resulting cross-linked materials were each comprised of thioether and ester linkages, with one retaining the hydrophilic phenols from magnolol, another having the phenols protected as an acetonide, and two others incorporating the phenols into additional cross-linking sites via hydrolytically labile carbonates or stable ether linkages. With this diversity of chemical compositions and structures, the thermosets displayed a range of thermomechanical properties including glass transition temperatures, Tg, 29-52 °C, onset of thermal degradation, Td, from about 290-360 °C, and ultimate strength up to 50 MPa. These tunable materials were studied in their degradation and biological properties with the aim of exploiting the antioxidant properties of the natural product. Hydrolytic degradation occurred under basic conditions (pH = 11) in all thermosets, but with kinetics that were dependent upon their chemical structures and mechanical properties: 20% mass loss was observed at 5, 7, 27, and 40 weeks for the thermosets produced from magnolol directly, acetonide-protected magnolol, bis(allyl carbonate)-functionalized magnolol, and bis(allyl ether)-functionalized magnolol, respectively. Isolated degradation products and model compounds displayed antioxidant properties similar to magnolol, as determined by both UV-vis and in vitro reactive oxygen species (ROS) assays. As these magnolol-based thermosets were found to also allow for extended cell culture, these materials may serve as promising degradable biomaterials.

Bio-based polycarbonates derived from the neolignan honokiol

Honokiol, a highly functional phenolic- and alkenyl-containing neolignan natural product isolated from several Magnolia plant species, is an interesting bio-based resource, which is shown to be useful directly as a monomer for the rapid and scalable synthesis of poly(honokiol carbonate) (PHC). PHC was synthesized in one step from the natural product using condensation polymerization methods. Polymers of number average molecular weight (Mn) ranging from 10–55 kDa were obtained on gram scales in yields up to 80%. Thermal analysis demonstrated high thermal stability, with degradation temperatures in excess of ca. 450 °C. Mechanical testing of several PHC polymers indicated a generally increasing storage modulus with increasing Mn and a similar trend with Tg. With an interest toward cardiovascular applications, initial cytotoxicity and fluorescence cell imaging studies were conducted and showed no cytotoxicity toward coronary venular endothelial cells (CVECs), which proliferated on PHC thin films up to a month. Bulk PHC is a robust material, as it underwent slow hydrolytic degradation under basic conditions (ca. 0.1% per day under 1 M NaOH(aq)), and no observable degradation under acidic and neutral conditions, each at 37 °C over 130 days. These polycarbonates serve as potential specialty engineering- or bio-materials derived from a commercially-available natural product monomer.

Effect of Tensile Stress on Smooth Muscle Cells

Cellular responses to mechanical stresses play an important role in the physiology of many cell types in healthy and diseased states. In vascular smooth muscle cells (VSMC), as in any other cells anchored to extracellular matrix, external mechanical stresses are imposed on a preexisting force equilibrium generated by the cytoskeletal tension. The ability to measure real-time mechanosensitive events at sub-cellular level in response to discrete and physiologically relevant mechanical stimulation is the critical component in understanding mechanically-induced cellular remodeling. Mechanical perturbation of a VSMC by the atomic force microscope (AFM) tip mimics the tensile stress in the vessel wall. By integrating tensile stress stimulation with simultaneous optical imaging using total internal reflection fluorescence and fast spinning-disk confocal microscopy, we have the unique opportunity of investigating in real-time the mechanically-induced cross-talk between apical and basal cell surface. Thus, we were able to measure vinculin and actin recruitment at focal adhesions upon tensile stress stimulation, and stress fibers bundling and reassembly. Understanding the real-time contractile and adhesion events associated with live VSMC response to force, provides fundamental new information regarding the coordinated cellular responses involved in VSMC adaptation to the local extracellular environment in the vessel wall.

Mechanically Induced Cell Signaling Stimulates Real-Time Cytoskeleton Remodeling

External mechanical stresses alter the structural and functional properties of the cells, leading to rapid responses that induce adaptive changes to the external environment. The extracellular matrix is responsible for a complex cross-talk needed for transmitting environmental signals to the cell through the focal adhesions as mediators of the process. An Atomic Force Microscope (AFM) probe functionalized with fibronectin was able to mechanically stimulate the apical surface of a live smooth muscle cell inducing significant changes in cell shape that can be recorded in real time by optical imaging. Due to the strong focal adhesion formed around the AFM tip, the cytoskeletal elements are directly manipulated through a matrix-integrin-actin linkage between the cell and the fibronectin coated tip. Following each controlled upward movement of the cantilever, the cell responds by presenting a biphasic change in height dependent of the treatment applied, and independent of time. In the same time, the cell reinforces its attachment to the substrate to better resist the mechanical stimulation by increasing focal adhesion and actin area at the basal cell level. Our measurements showed significant differences between control cells and cells where the intracellular tension was modulated by RhoA. Thus, in cells transfected with RhoA constitutively active the cell reactive-response presents higher amplitude than control because the cell is stronger due to the presence of more actin fibers. A different response was found when cells were transfected with RhoA dominant negative, which decreases intracellular tension, such that actin filaments are present only at the cell boundaries. Under these conditions, at the same force level, the AFM tip detaches from the apical cell surface. These innovative approaches offer new information for understanding live cell remodeling and dynamics in response to mechanical force.