Synthesis of a photo-caged aminooxy alkane thiol

A photo-caged aminooxy alkane thiol synthesized in 7 steps and 15% overall yield was used to form a self-assembled monolayer (SAM). Photo-deprotection on the surface was confirmed by FT-IR spectroscopy and contact angle goniometry. Conjugation of a small molecule ketone, ethyl levulinate, further confirmed the presence of aminooxy groups on the surface

Site-specific protein immobilization through N-terminal oxime linkages

Immobilizing proteins in specific orientations is important for diagnostic protein arrays, biomaterials, and other applications where retention of bioactivity is essential. We report an approach for protein micropatterning that exploits a chemoselective reaction to conjugate proteins at the N-terminus to polymer films. A copolymer from 2-hydroxyethyl methacrylate and a Boc-protected aminooxy tetra(ethylene glycol) methacrylate was synthesized by radical polymerization. Boc groups were locally deprotected using photoacid generator-based photolithography. Micropatterns were verified by fluorescence microscopy utilizing green fluorescent aldehyde microspheres. Streptavidin that was subjected to a transamination reaction to install an -ketoamide group at the N-terminus was conjugated to the patterns by oxime bond formation. Since the majority of proteins may be modified to contain a reactive carbonyl group, this methodology should be applicable to pattern a wide variety of proteins specifically through the N-terminus

Chemoselective Immobilization of Proteins by Microcontact Printing and Bio-orthogonal Click Reactions

Herein, a combination of microcontact printing of functionalized alkanethiols and site-specific modification of proteins is utilized to chemoselectively immobilize proteins onto gold surfaces, either by oxime- or copper-catalyzed alkyne-azide click chemistry. Two molecules capable of click reactions were synthesized, an aminooxy-functionalized alkanethiol and an azide-functionalized alkanethiol, and self-assembled monolayer (SAM) formation on gold was confirmed by IR spectroscopy. The alkanethiols were then individually patterned onto gold surfaces by microcontact printing. Site-specifically modified proteinshorse heart myoglobin (HHMb) containing an N-terminal -oxoamide and a red fluorescent protein (mCherry-CVIA) with a C-terminal alkynewere immobilized by incubation onto respective stamped functionalized alkanethiol patterns. Pattern formation was confirmed by fluorescence microscopy

Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly­(methacrylamidotrehalose) or “poly­(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (<i>n</i> = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P­(AEMA) and P­(AEMA)]. First, we demonstrate that the poly­(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly­(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly­(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly­(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC₅₀ of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly­(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications

Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly­(methacrylamidotrehalose) or “poly­(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (<i>n</i> = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P­(AEMA) and P­(AEMA)]. First, we demonstrate that the poly­(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly­(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly­(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly­(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC₅₀ of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly­(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications

Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT₄₃. The pMAT₄₃ macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-<i>b</i>-AEMA cationic-block copolymers, pMAT-<i>b</i>-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-<i>b</i>-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-<i>b</i>-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo

High-Throughput Excipient Discovery Enables Oral Delivery of Poorly Soluble Pharmaceuticals

Polymeric excipients are crucial ingredients in modern pills, increasing the therapeutic bioavailability, safety, stability, and accessibility of lifesaving products to combat diseases in developed and developing countries worldwide. Because many early-pipeline drugs are clinically intractable due to hydrophobicity and crystallinity, new solubilizing excipients can reposition successful and even failed compounds to more effective and inexpensive oral formulations. With assistance from high-throughput controlled polymerization and screening tools, we employed a strategic, molecular evolution approach to systematically modulate designer excipients based on the cyclic imide chemical groups of an important (yet relatively insoluble) drug phenytoin. In these acrylamide- and methacrylate-containing polymers, a synthon approach was employed: one monomer served as a precipitation inhibitor for phenytoin recrystallization, while the comonomer provided hydrophilicity. Systems that maintained drug supersaturation in amorphous solid dispersions were identified with molecular-level understanding of noncovalent interactions using NOESY and DOSY NMR spectroscopy. Poly­(<i>N</i>-isopropylacrylamide-<i>co</i>-<i>N</i>,<i>N</i>-dimethylacrylamide) (poly­(NIPAm-<i>co</i>-DMA)) at 70 mol % NIPAm exhibited the highest drug solubilization, in which phenytoin associated with inhibiting NIPAm units only with lowered diffusivity in solution. In vitro dissolution tests of select spray-dried dispersions corroborated the screening trends between polymer chemical composition and solubilization performance, where the best NIPAm/DMA polymer elevated the mean area-under-the-dissolution-curve by 21 times its crystalline state at 10 wt % drug loading. When administered to rats for pharmacokinetic evaluation, the same leading poly­(NIPAm-<i>co</i>-DMA) formulation tripled the oral bioavailability compared to a leading commercial excipient, HPMCAS, and translated to a remarkable 23-fold improvement over crystalline phenytoin