Drug Discovery : Drug delivery


Enhancing Cell Penetration to Improve Drug Delivery

UW–Madison researchers have developed a method for enhancing cellular uptake of a cargo molecule by covalently bonding fluorenyl groups to it. The fluorenylated molecule is then contacted with the cell or tissue. Cellular uptake may be in vivo or in vitro and includes at least partial penetration into the cytosol.

Bioreversible Boronates Improve Drug Delivery

A UW–Madison researcher has developed methods and reagents for enhancing cellular uptake in vivo or in vitro by attaching to any desired molecule one or more phenylboronic acid groups. The method is bioreversible; the boronate compound is cleaved from the molecule by intracellular enzymes, leaving its ‘cargo’ unaltered.

Advantageously, boronic acids readily form esters within the dense forest of polysaccharides, known as the glycocalyx, found on the surface of many cells. Targeting therapeutic agents to the glycocalyx has been shown to enhance cellular delivery. In addition, boronate groups are compatible with human physiology, appearing in chemotherapeutic agents and other remedies.

Lipid-Free Emulsions for Delivering Anesthesia, Other Hydrophobic Drugs

UW–Madison researchers have developed non-lipid nanoemulsions for delivering propofol and other hydrophobic compounds. The formulations contain miniscule droplets of semifluorinated block copolymers and phospholipid surfactants, and are highly stable without the need for conventional lipid components like soybean oil.

The ingredients can be adjusted to (i) enhance stability, (ii) accelerate or slow drug release rates and (iii) increase shelf life.

Lipid-Free, Stabilized Emulsions for Delivering Anesthesia and Other Hydrophobic Drugs

UW–Madison researchers have developed non-lipid nanoemulsions for delivering propofol and other hydrophobic compounds. The formulations contain miniscule droplets of semifluorinated block copolymers and perhalogenated fluorous compounds, such as perfluorooctyl bromide or perfluorodecalin.

These ingredients are capable of forming a stable nanoemulsion without the need for conventional lipid components (e.g., soybean oil) that support bacterial and/or fungal growth. The emulsions have enhanced stability with respect to droplet size due to decreased particle coarsening, coagulation and/or phase separation.

Blood-Brain Barrier Targeting Antibodies to Improve Drug Delivery

UW–Madison researchers have identified a pair of single-chain antibody fragments (scFv15 and scFv38) that may help drugs cross the BBB. The two promising new antibodies are capable of binding antigens expressed at the BBB in vivo.

The researchers panned a human scFv library to identify candidates that specifically bind to brain endothelial cell receptors and may pass through the BBB. Drugs or drug carriers could be attached to these fragments and then transported into the brain.

Thermogel for Combination Drug Delivery

UW–Madison researchers have developed hydrogels for delivering drug combinations to cancer patients. The gel is made of a solution of heat-sensitive, biodegradable block copolymers (PLGA-PEG-PLGA) that turn semisolid at body temperature.

The gel can contain a combination of therapeutic agents like rapamycin, paclitaxel and 17-AAG. After being administered to a patient, the gel releases the drugs at a controlled rate, and then biodegrades into nontoxic fragments.

Improving Drug Delivery with Boronic Acids

UW–Madison researchers have developed methods for boronating cargo molecules to mediate their entry into mammalian cells via the glycocalyx. ‘Cargo’ molecules include drugs, proteins, labels, amino acids or any other desired molecule.

Boronation methods include ligating, crosslinking or otherwise bonding phenylboronic acids/oligopeptides to the cargo molecule. It is believed that the boronates undergo complexation with glycans on the cell surface. This facilitates the molecule’s entry into cell endosomes, where the cargo is released by enzyme action.

Lipogel for Drug Delivery

UW–Madison researchers have developed a liposome-encapsulated hydrogel, or lipogel, that provides high loading and sustained release of drugs.

Hydrogel precursors are encapsulated into liposomes, which then are extruded, treated accordingly and polymerized to yield lipogels. The lipogel then can be loaded with a desired drug through the use of a pH gradient.

Biocompatible Formulations of Poorly Soluble Anticancer Drugs Such as Gossypol

UW–Madison researchers have developed biocompatible micelles loaded with gossypol or combinations of gossypol and other anticancer drugs such as paclitaxel, 17-AAG and cyclopamine. These drug formulations are stable and provide improved bioavailability without causing toxicity. They enable the intravenous delivery of cancer therapeutics like gossypol that are poorly soluble in water.

Multilayered Films for the Controlled Release of Anionic Molecules, Including Nucleic Acids

UW–Madison researchers now have developed PEMs that incorporate cationic charge shifting polymers to promote the controlled release of multiple different anionic molecules with distinct release profiles.  The use of these charge dynamic polymers enables more sophisticated and tunable control of biomolecule release than other types of degradable layers and allows the fabrication of ultrathin films that can sustain the release of nucleic acids for variable periods of time ranging from several days to several weeks or months.  For example, a single multilayered film could be used for the rapid, short-term release of a first DNA construct followed by the long-term release of a second, different DNA construct.

An Injectable Nanovehicle for a Cancer Treatment Combination of Paclitaxel, Rapamycin and 17-AAG

A UW-Madison inventor has developed a novel, nontoxic nanoformulation of multiple anticancer agents.  This nanoformulation consists of rapamycin, paclitaxel and 17-AAG, encapsulated by safe PEG-b-PLA micelles. 

These polymeric micelles can be used to safely and effectively deliver multiple active agents, including therapeutics that are poorly soluble in water, enabling the simple, sterile and synergistic delivery of paclitaxel, rapamycin and 17-AAG.  The combination of paclitaxel, rapamycin and 17-AAG is particularly effective because it targets the PI3-AKT-mTOR pathway, a common deregulated pathway in cancer, leading to disruption at both AKT and mTOR.

Novel Charge Shifting Anionic Polymers for the Controlled Release of Cationic Agents from Surfaces

UW-Madison researchers now have developed novel compositions and methods for creating charge shifting anionic polymers.  These polymers could be used to create polymeric multilayers and thin films that can controllably release cationic agents such as proteins and peptides. 

The anionic polymers are prepared by the reaction of small-molecule anhydrides, such as citraconic anhydride, with primary amine side chains on a polymer backbone, such as poly(allylamine hydrochloride), a weak polyelectrolyte widely used for the fabrication of polyelectrolyte multilayers.  This reaction yields an anionic, carboxylate-functionalized polymer that can undergo a dynamic change in charge state (from anionic to less anionic) to trigger the “unpackaging” of cationic molecules.

High Throughput Assay for Sugar-Mediated Drug Transport

UW-Madison researchers have developed a systematic platform for rapidly assessing the ability of a diverse range of sugars to enhance the uptake and selectivity of sugar conjugates. This invention may lead to the discovery of sugar molecules that improve the delivery of cancer therapeutics.

First, glycorandomization (see WARF reference numbers P04020US and P04455US) is used to generate a library of molecules that differ only by the sugars attached. Then each of these glycosylated molecules is contacted with cells, and their uptake into the cells is assessed relative to that of a corresponding molecule without the attached sugars. To determine selectivity, these sugar conjugates can be contacted with cells from different cell lines and their uptake compared to see if it is elevated in cells from a particular line.

Using this strategy, the researchers found that slight changes in sugar structure can lead to drastic changes in in vitro cellular uptake. They identified sugars that impart up to an eight-fold increase in selective uptake by tumor, rather than normal, cell lines. They also identified sugars that provide greater than a 10-fold increase in uptake as compared to the conventional sugars glucose, 2-deoxyglucose or FDG.

Layer-by-Layer Covalent Assembly of Reactive Ultrathin Films

UW–Madison researchers have developed robust methods for the layer-by-layer fabrication of covalently cross-linked ultrathin films.  This approach makes use of fast and efficient “click”-type interfacial reactions between poly(2-alkenyl azlactone)s and appropriately functionalized polyamines.  In contrast to conventional, aqueous methods for the layer-by-layer fabrication of thin films, fabrication of these ultrathin films occurs in organic solvents and is driven by rapid formation of covalent bonds during assembly. This approach also yields films with residual azlactone groups that can be used to tailor the surface properties of the films by treatment with a broad range of chemical and biological functionalities.

Hydrogel Drug Delivery Device as an Alternative to Pressurized Gas or Voltage Transdermal Technology

UW-Madison researchers have developed a drug delivery device that provides a controlled infusion of a drug to an individual.  The device includes a reservoir that holds the drug.  A predetermined stimulus, which may be activated by the individual, causes a hydrogel to exert pressure on the reservoir, dispensing the drug.

Coatings That Inhibit Crystallization of Amorphous Drugs to Improve Stability

UW-Madison researchers have developed a method of coating amorphous drugs to inhibit surface crystallization and improve their stability.  An ultra thin polyelectrolyte coating or other biocompatible immobilizing material is applied to the surface of an amorphous solid.  This coating allows amorphous pharmaceuticals to maintain their amorphous state, and therefore their solubility, over extended periods of time. 

Efficient Method of Producing Glass with Enhanced Stability

UW-Madison researchers have developed an efficient method of producing extremely stable amorphous material through vapor deposition of organic molecules. The key to their method involves controlling the substrate temperature used for film deposition to 50 K below the glass transition temperature (Tg) of the substance being deposited and depositing the material at a rate of less than 1 nm/s.

Current instrumentation used for vapor deposition typically does not provide a means of controlling the temperature of the substrate. As a result, the substrate generally stays at room temperature, which is not optimal for producing stable glasses. Film layers deposited on these relatively cold substrates “freeze” in place upon deposition with no opportunity to form a stable glass.

The inventors modified their instrument to provide a means of controlling the temperature of the substrate. When the substrate temperature is controlled to about 50 K below Tg and the material in each layer is deposited at a relatively slow rate, the molecules at the surfaces of each layer are mobile enough to pack efficiently with each other, creating a more integrated and therefore more stable glass film. 

Multilayered Film for Delivering Proteins and Other Small Molecules into Cells

UW-Madison researchers have developed a new way of delivering proteins and other small molecules into cells. This approach uses a cationic “anchor” to improve incorporation of proteins into multilayered films.

Before the protein or small molecule is integrated into the film, a cationic protein transduction domain, such as nonaarginine, is attached to it. Appending short, cationic peptides or oligomers to proteins can facilitate their layer-by-layer assembly into PEMs, as well as their uptake by cells.

Then the cationic molecule is incorporated into a polyelectrolyte multilayered film, along with anionic polymers such as sodium polystyrene sulfonate, to result in a multilayered assembly that is preferably about 80 nanometers thick. When this composition is presented to a cell, the film dissolves, delivering the molecule to the cell.

Improved Micellar Delivery System for Hydrophobic or Fluorophilic Drugs

UW-Madison researchers have created highly stable and biocompatible micelles for the delivery of hydrophobic or fluorinated therapeutic agents. These micelles are self-assembled from semi-fluorinated copolymers consisting of discrete hydrophilic, fluorophilic and hydrophobic domains. Specifically, the copolymers may include blocks of polyethylene glycol, fluorocarbon and phospholipid.

Encapsulating hydrophobic and/or fluorophilic compounds with these micelles provides enhanced solubilization, protection and stabilization as compared to conventional drug delivery methods. The fluorophilic block effectively seals the hydrophobic core, making the micelles and therapeutic agents more stable, and can be modified to selectively “tune” the release rate of the encapsulated compound.  

Nanoparticles That Target Dendritic Cells

UW-Madison researchers have developed a system for delivering vaccines and other biomolecules to dendritic cells. This system includes carbon nanoparticles that are preferentially taken up by dendritic cells, rather than macrophages.

Antigens, dendritic cell-targeting antibodies and dendritic cell-activating substances may be attached to the nanoparticles. The antigens are capable of inducing a specific T-cell response and can be associated with infectious disease or a tumor. When delivered to dendritic cells, these nanoparticles enhance immune response.

Other biomolecules, including targeting compounds, therapeutic agents and detectable labels, can be attached to the nanoparticles as well. Targeting compounds may be attached to enhance the uptake of the nanoparticles by dendritic cells. To treat autoimmune diseases, a cytotoxic agent could be attached to the nanoparticles to selectively target and kill aberrant dendritic cells. Fluorescent or radioactive labels could be added to make it easier to isolate dendritic cells. 

Fluoropolymer-Based Emulsions for the Intravenous Delivery of Fluorinated Volatile Anesthetics

UW-Madison researchers have developed a novel formulation for the intravenous delivery of fluorinated volatile anesthetics. The formulation consists of an aqueous solution; semi-fluorinated block copolymers with hydrophilic and fluorophilic blocks; a fluorinated therapeutic compound, such as a volatile anesthetic; and a stabilizing additive. The formulation may be an emulsion, with the block copolymers, therapeutic compound and stabilizing additive dispersed in the aqueous solution. The block copolymers encapsulate large amounts of the therapeutic compound in micelles or other supramolecular structures that can circulate in the blood and deliver the compound to target tissues.

As compared to conventional lipid-based delivery systems, this formulation provides enhanced performance. When injected intravenously, it can rapidly and efficiently deliver high concentrations of fluorinated volatile anesthetics to specific active sites on ion channels and neurotransmitter receptors.

Blood-Brain Barrier Targeting Antibodies

UW-Madison researchers have identified several single-chain antibody fragments (scFv) that may provide a targeting mechanism to help drugs cross the blood-brain barrier. They mined a human scFv library to identify scFv that specifically bind to brain endothelial cell receptors and may pass through the blood-brain barrier. Drugs or drug carriers could be attached to these fragments and then transported into the brain.

Additionally, the researchers discovered that one scFv, known as scFvJ, binds to an antigen that has been identified as the neural cell adhesion molecule (NCAM), a known endocytosing receptor.  NCAM may play a role in cell adhesion, synaptic plasticity and learning and memory.

Elastin-Like Biopolymers as Delivery Vehicles for Gene Therapy

UW-Madison researchers have developed IBNs capable of actively targeting therapeutic nucleic acids, or other negatively charged molecules, to specific cells or tissues.  Positively charged amino acid residues that make up the polycationic region within the IBN platform electrostatically condense the negatively charged, polyanionic nucleic acids into an electroneutral core, where they are protected from degradation.  Targeting modalities, such as ligands, monoclonal antibodies or single chain variable fragment antibodies, are linked to this core by the ELP sequence.

Multilayer Tissue Regeneration System

A UW-Madison researcher has developed an approach for regenerating natural skeletal tissues that more closely mimics in vivo conditions by localizing and temporally controlling the activity of multiple growth factors. This method for growing tissue is based on a matrix of minerals and growth factors. Engineered protein growth factors are incorporated into the layers of the inorganic matrix. Each layer is designed to dissolve at a separate rate. As the matrix material gradually breaks down, the growth factors are delivered sequentially, enabling the growth of new bone tissue. Alternatively, the growth factors can be engineered to bind to the surface of the inorganic matrix.

Localized Delivery of Nucleic Acid by Polyelectrolyte Assemblies

UW-Madison researchers have developed ultrathin, multilayered polyelectrolyte films that permit the localized delivery of nucleic acids to cells from the surfaces of implantable materials. To form the polyelectrolyte coating, nucleic acids and polycations are deposited layer-by-layer onto the surface of an implantable device. After implantation, nucleic acids are delivered only to the cells in direct contact with the device surface. The polyelectrolyte film promotes the direct and self-sufficient transfection of those cells to enable local production of therapeutic agents.

Spatial Control of Signal Transduction

UW-Madison researchers have developed a novel approach for the delivery of growth factors and other biologically active signaling molecules that allows for spatial control of signal transduction. Molecular handles (e.g., peptides or oligonucleotides) with an affinity for growth factors or other signaling molecules are attached to specific locations on a surface or within a matrix. The surface or matrix includes a translucent, biologically inert polymer backbone. Signaling molecules are drawn to the precisely desired location of the substrate or matrix by associating specifically, reversibly and non-covalently with the handles. The affinity of the molecular handle for the signaling molecule is tailored so that it selectively attracts the growth factor but does not bind so tightly that it blocks interaction of the growth factor with percursor cells. The signaling molecules can then be presented to precursor cells in locally high concentrations, mimicking complex developmental processes like organogenesis, which is particularly important in stem cell-based approaches to tissue regeneration.

Micelle Composition with Enhanced Drug Loading Capacity and Stability

UW-Madison researchers have developed formulations for efficiently loading hydrophobic compounds into polymeric micelles.  Adding alpha-tocopherol (vitamin E) as an excipient enhances the drug loading capacity and stability of the micelles, possibly by modifying their core properties.  PEG-DPSE micelles containing rapamycin normally come out of solution in less than two hours; however, when tocopherol is added, the micelles are stable for several days.  The addition of tocopherol also increases the loading of rapamycin into the micelles more than three-fold.  Similar results have been obtained with novel analogs of the HSP 90 inhibitor geldanamycin.  

Fusion Proteins for Targeted Delivery of Chemotherapeutic Agents

A UW-Madison researcher has developed fusion proteins that act as targeted drug carriers. The proteins are derived from molecules, such as apoproteins, that possess natural drug-binding capabilities. They are engineered to contain cell-targeting peptides and may also exhibit altered drug-binding characteristics.

Chemotherapeutic agents or other drugs are complexed with the fusion proteins so that the drugs can be delivered directly to a specific location in the body, such as a tumor site. For example, C-1027 is a potent anticancer antibiotic that is composed of an apoprotein, encoded by the cagA gene, bound to an enediyne chromophore. A tumor cell surface-specific peptide or a tumor vasculature-specific peptide can be fused to the CagA apoprotein to deliver C-1027 to the tumor site.

Charge-Dynamic Polymers for Delivering Anionic Compounds, Such as DNA, into Cells

UW-Madison researchers have developed polymers that allow temporal control over the dissociation of DNA from polymer/DNA interpolyelectrolyte complexes. The cationic polymers undergo dynamic changes in charge states (from cationic to less cationic) to trigger the “unpackaging” of anionic molecules from IPECs. The polymers possess cationic charge densities that result from the number, type, and position of functional groups attached to the backbone; specifically, cationic charge densities decrease when one or more of the functional groups is removed.

In one embodiment, side chain esters are introduced to linear poly(ethylene imine) (PEI) via conjugate addition chemistry. The PEI is then complexed with an anionic molecule such as DNA. When the pendant ester groups are hydrolyzed, the cationic charge density of the polymer is reduced, promoting the dissociation of the polymer/DNA complex and efficient release of DNA.

Microfluidic Device for Drug Delivery

As an alternative to oral administration, UW-Madison researchers have developed a microfluidic device for delivering a steady infusion of a drug through the skin. The device may take the form of a thin, transcutaneous patch that can be worn for extended periods of time. The device includes a reservoir for storing the drug, and a valve that connects the reservoir to an output needle inserted into the patient’s skin. A pressure source causes the drug to flow from the reservoir to the needle. The key advantage of this design is that the valve can move between the open and closed positions in response to a predetermined condition in the patient’s physiological fluids, providing autonomous control of drug flow.

Engineering of Ribonuclease Zymogens

UW-Madison researchers have developed a method of engineering an enzyme into a zymogen. They converted the ribonuclease enzyme RNase A into a zymogen by adding a bridge of amino acids that link the amino and carboxyl termini of the enzyme. The bridge contains a protease cleavage site specific to a certain protease. RNase A can be made cytotoxic; thus, this technology suggests how a cytotoxic enzyme, engineered in the form of a zymogen, could be delivered in an inactive state and then activated only when in the presence of a protease inside a particular target cell.