Andreas M. Nyström

Applications of Orthogonal, “Click” Chemistries in the Synthesis of Functional Soft Materials

Department of Chemistry, Department of Radiology, Washington University in Saint Louis, Saint Louis, Missouri 63130, Department of Chemistry, Texas A&M University, College Station, Texas 77842, Cancer Center Karolinska, Department of Oncology-Pathology CCK, R8:03 Karolinska Hospital and Institute, SE-171 76 Stockholm, Sweden, and Department of Chemistry and Biochemistry, Department of Materials, and Materials Research Laboratory, University of California, Santa Barbara, California 93106

One-pot Synthesis of Metal-Organic Frameworks with Encapsulated Target Molecules and Their Applications for Controlled Drug Delivery.

Many medical and chemical applications require target molecules to be delivered in a controlled manner at precise locations. Metal-organic frameworks (MOFs) have high porosity, large surface area, and tunable functionality and are promising carriers for such purposes. Current approaches for incorporating target molecules are based on multistep postfunctionalization. Here, we report a novel approach that combines MOF synthesis and molecule encapsulation in a one-pot process. We demonstrate that large drug and dye molecules can be encapsulated in zeolitic imidazolate framework (ZIF) crystals. The molecules are homogeneously distributed within the crystals, and their loadings can be tuned. We show that ZIF-8 crystals loaded with the anticancer drug doxorubicin (DOX) are efficient drug delivery vehicles in cancer therapy using pH-responsive release. Their efficacy on breast cancer cell lines is higher than that of free DOX. Our one-pot process opens new possibilities to construct multifunctional delivery systems for a wide range of applications.

Safety assessment of nanomaterials: implications for nanomedicine.

Nanotechnologies offer exciting opportunities for targeted drug delivery which is anticipated to increase the efficacy of the drug and reduce potential side-effects, through the reduction of the dose of the drug in bystander tissues and an increase of the drug at the desired target site. Nevertheless, understanding whether the nano-scale carriers themselves may exert adverse effects is of great importance. The small size may enable nanoparticles to negotiate various biological barriers in the body which could, in turn, give rise to unexpected toxicities. On the other hand, the potential of nanoparticles to cross barriers can also be exploited for drug delivery. Determining the fate of nanoparticles following their therapeutic or diagnostic application is critical: are nanoparticles excreted, or biodegraded, or do they accumulate, potentially leading to harmful long-term effects? The bio-corona of proteins or lipids on the surface of nanoparticles is a key parameter for the understanding of biological interactions of nanoparticles. In the present review, we discuss some of the major challenges related to safety of nanomedicines.

The importance of chemistry in creating well-defined nanoscopic embedded therapeutics: devices capable of the dual functions of imaging and therapy.

Nanomedicine is a rapidly evolving field, for which polymer building blocks are proving useful for the construction of sophisticated devices that provide enhanced diagnostic imaging and treatment of disease, known as theranostics. These well-defined nanoscopic objects have high loading capacities, can protect embedded therapeutic cargo, and offer control over the conditions and rates of release. Theranostics also offer external surface area for the conjugation of ligands to impart stealth characteristics and/or direct their interactions with biological receptors and provide a framework for conjugation of imaging agents to track delivery to diseased site(s). The nanoscopic dimensions allow for extensive biological circulation. The incorporation of such multiple functions is complicated, requiring exquisite chemical control during production and rigorous characterization studies to confirm the compositions, structures, properties, and performance. We are particularly interested in the study of nanoscopic objects designed for treatment of lung infections and acute lung injury, urinary tract infections, and cancer. This Account highlights our work over several years to tune the assembly of unique nanostructures. We provide examples of how the composition, structure, dimensions, and morphology of theranostic devices can tune their performance as drug delivery agents for the treatment of infectious diseases and cancer. The evolution of nanostructured materials from relatively simple overall shapes and internal morphologies to those of increasing complexity is driving the development of synthetic methodologies for the preparation of increasingly complex nanomedicine devices. Our nanomedicine devices are derived from macromolecules that have well-defined compositions, structures, and topologies, which provide a framework for their programmed assembly into nanostructures with controlled sizes, shapes, and morphologies. The inclusion of functional units within selective compartments/domains allows us to create (multi)functional materials. We employ combinations of controlled radical and ring-opening polymerizations, chemical transformations, and supramolecular assembly to construct such materials as functional entities. The use of multifunctional monomers with selective polymerization chemistries affords regiochemically functionalized polymers. Further supramolecular assembly processes in water with further chemical transformations provide discrete nanoscopic objects within aqueous solutions. This approach echoes processes in nature, whereby small molecules (amino acids, nucleic acids, saccharides) are linked into polymers (proteins, DNA/RNA, polysaccharides, respectively) and then those polymers fold into three-dimensional conformations that can lead to nanoscopic functional entities.

Stability and biocompatibility of a library of polyester dendrimers in comparison to polyamidoamine dendrimers

Dendrimers can be designed for several biomedical applications due to their well-defined structure, functionality and dimensions. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5-2 nm. In addition, dendrimers with two different chemical surfaces (neutral with hydroxyl end group and anionic with carboxylic end group) and dendrons corresponding to the structural fragments of the dendrimers were evaluated. Commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group were also included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differentiated into macrophage-like cells as well as in primary human monocyte-derived macrophages. Excellent biocompatibility was observed for the entire hydroxyl functional bis-MPA dendrimer library, whereas the cationic, but not the neutral PAMAM exerted dose-dependent cytotoxicity in cell lines and primary macrophages. Studies to evaluate material stability as a function of pH, temperature, and time, demonstrated that the stability of the 4th generation hydroxyl functional bis-MPA dendrimer increased at acidic pH. Taken together, bis-MPA dendrimers are degradable and non-cytotoxic to human cell lines and primary cells.

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles.

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.

19F- and Fluorescently Labeled Micelles as Nanoscopic Assemblies for Chemotherapeutic Delivery

Micelles from amphiphilic star-block copolymers, having a hydrophobic hyperbranched core and amphiphilic fluoropolymer arms, were constructed as drug delivery agent assemblies. A series of polymer structures was constructed from consecutive copolymerizations of 4-chloromethylstyrene with dodecyl acrylate and then 1,1,1- trifluoroethyl methacrylate with tert-butyl acrylate, followed by acidolysis to release the hydrophilic acrylic acid residues. These structures were labeled with cascade blue as a fluorescence reporter. The series of materials differed primarily in the ratio of 1,1,1-trifluoroethyl methacrylate to acrylic acid units, to give differences in fluorine loading and hydrophobicity/hydrophilicity balance. Doxorubicin (DOX) was used as a therapeutic to study the loading, release, and cytotoxicity of these micellar constructs on an U87-MG-EGFRvIII-CBR cell line. The micelles, with TEM-measured diameters ranging 5-9 nm and DLS-measured hydrodynamic diameters 20-30 nm, had loading capacities of ca. 4 wt % of DOX. The DOX-loaded micelles exhibited potent cytotoxicity with cell viabilities of 60-25% at 1.0 microg/mL effective DOX concentrations, depending upon the polymer composition, as determined by MTT assays. These cell viability values are comparable to that of free DOX, suggesting an effective release of the cargo and delivery to the cell nuclei, which was further confirmed by fluorescence microscopy of the cells. 19F-NMR spectroscopy indicated a partial degradation of the surface-available trifluoroethyl ester linkages of the micelles, which may have accelerated the release of DOX. 19F-NMR spectroscopy was also employed to confirm and to quantify the cell uptake of the micelles. These dual fluorescent- and 19F-labeled and chemically functional micelles may be used potentially in a variety of applications, such as cell labeling, imaging, and therapeutic delivery.

Poly(ethylene glycol)-Based Thiol-ene Hydrogel Coatings−Curing Chemistry, Aqueous Stability, and Potential Marine Antifouling Applications

Photocured thiol-ene hydrogel coatings based on poly(ethylene glycol) (PEG) were investigated for marine antifouling purposes. By varying the PEG length, vinylic end-group, and thiol cross-linker, a library of hydrogel coatings with different structural composition was efficiently accomplished, with or without ester linkages. The thiol-methacrylate and thiol-allyl systems were evaluated with respect to curing, degradation, as well as antifouling properties. Methacrylate-based systems exhibited homopolymerization, whereas allyl-based systems reacted more selectively through thiol-ene couplings reaction. The ester-free hydrogels elucidated higher hydrolytic stability whereas longer PEG chains accelerated the degradation process. The antifouling properties were evaluated by protein adsorption with Bovine serum albumin (BSA) and bioassays with the marine bacteria, Cobetia marina, and the marine diatom, Amphora coffeaeformis; in all tests, longer PEG lengths improved the antifouling properties.

Tuning core vs. shell dimensions to adjust the performance of nanoscopic containers for the loading and release of doxorubicin

Detailed studies were performed to probe the effects of the core and shell dimensions of amphiphilic, shell crosslinked, knedel-like polymer nanoparticles (SCKs) on the loading and release of doxorubicin (DOX), a widely-used chemotherapy agent, in aqueous buffer, as a function of the solution pH. Effects of the nanoparticle composition were held constant, by employing SCKs constructed from a single type of amphiphilic diblock copolymer, poly(acrylic acid)-b-polystyrene (PAA-b-PS). A series of four SCK nanoparticle samples, ranging in number-average hydrodynamic diameter from 14-30 nm, was prepared from four block copolymers having different relative block lengths and absolute degrees of polymerization. The ratios of acrylic acid to styrene block lengths ranged from 0.65 to 3.0, giving SCKs with ratios of shell to core volumes ranging from 0.44 to 2.1. Although the shell thicknesses were calculated to be similar (1.5-3.1 nm by transmission electron microscopy (TEM) calculations and 3.5-4.9 nm by small angle neutron scattering (SANS) analyses), two of the SCK nanoparticles had relatively large core diameters (19±2 and 20±2 nm by TEM; 17.4 and 15.3 nm by SANS), while two had similar, smaller core diameters (11±2 and 13±2 nm by TEM; 9.0 and 8.9 nm by SANS). The SCKs were capable of being loaded with 1500-9700 DOX molecules per each particle, with larger numbers of DOX molecules packaged within the larger core SCKs. Their shell-to-core volume ratio showed impact on the rates and extents of release of DOX, with the volume occupied by the poly(acrylic acid) shell relative to the volume occupied by the polystyrene core correlating inversely with the diffusion-based release of DOX. Given that the same amount of polymer was used to construct each SCK sample, SCKs having smaller cores and higher acrylic acid vs. styrene volume ratios were present at higher concentrations than were the larger core SCKs, and gave lower final extents of release., Higher final extents of release and faster rates of release were observed for all DOX-loaded particle samples at pH 5.0 vs. pH 7.4, respectively, ca. 60% vs. 40% at 60 h, suggesting promise for enhanced delivery within tumors and cells. By fitting the data to the Higuchi model, quantitative determination of the kinetics of release was made, giving rate constants ranging from 0.0431 to 0.0540 h⁻¹/² at pH 7.4 and 0.106 to 0.136 h⁻¹/² at pH 5.0. In comparison, the non-crosslinked polymer micelle analogs exhibited rate constants for release of DOX of 0.245 and 0.278 h⁻¹/² at pH 7.4 and 5.0, respectively. These studies point to future directions to craft sophisticated devices for controlled drug release.

Linear dendritic polymeric amphiphiles with intrinsic biocompatibility: synthesis and characterization to fabrication of micelles and honeycomb membranes

Linear dendritic hybrid materials enable a range of architectural variations which offers novel possibilities in the tailoring of polymeric materials. In this study dendrons based on the 2,2-bis(methylol)propionic acid (bis-MPA) building block, bearing click chemistry moieties in the core and peripheral hydroxyl functionalities, have been used as macroinitiators for ring opening polymerization of e-caprolactone. A library of star branched polymers with poly(e-caprolactone) chains was initially constructed using dendrons up to 4th generation. In a second step, the popular CuAAC or thiol–ene click reaction was efficiently used to attach poly(ethylene glycol) chains of different lengths to the core. Potential applications of the resulted amphiphilic linear dendritic hybrids were investigated. Both self-assembled micelles loaded with doxorubicin anticancer drug and ordered honeycomb membranes with enhanced surface area were successfully fabricated and characterized.