Drug Discovery : Drug delivery


Polyplex Delivery System for Proteins, Nucleic Acids and Complexes

UW-Madison researchers have developed an improved biocompatible polyplex that can successfully deliver various biomolecular payloads (e.g., nucleic acids, RNP, RNP together with a single-stranded oligonucleotide DNA donor template). This polyplex contains two polymeric chains that can interact with negatively charged nucleic acid and/or protein payloads – a cationic polymer (poly-β-amino acid, PBAA) containing glutathione (GSH)-responsive disulfide bonds in the backbone and a second polymer that includes modifiable ends (polyethylene glycol, PEG) . Randomly incorporated imidazole groups in the cationic polymer enhance endosomal escape of the polyplex. To further enhance the stability of the polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) are conjugated to the polymers. The crosslinked polyplexes formed by host–guest interaction between β-CD and AD are more stable than non-crosslinked polyplexes in physiological conditions. Once in the cytosol, the disulfide bonds are cleaved by GSH, thereby releasing the protein/nucleic acid from the polyplex. Importantly, at the PEG terminal ends, simple chemistry can be used to add various cellular or tissue-specific targeting ligands and imaging probes.

When compared to a commercially available transfection reagent (Lipofectamine 2000), the polyplex significantly decreased toxicity and showed equal or better nucleic acid transfection efficiency in HEK 293 and RAW 264.7 cells. Testing both sgRNA/Cas9 complex (i.e., RNP) and RNP together with single-stranded DNA complex, the inventors showed that using the polyplex delivery system yielded superior gene knockout and gene editing results.

Nanocapsule Delivery System for Ribonucleoproteins

UW–Madison researchers have engineered a biodegradable GSH-responsive nanocoating surrounding the sgRNA/Cas9 RNP complex for efficient delivery into cells. The RNP nanocapsule is a polymeric network synthesized from a mixture of (meth)acrylate monomers, acrylate crosslinkers and acrylate polyethylene glycol (PEG), built around the RNP cargo. The interactions between the RNP and the monomers include electrostatic and hydrophobic interactions and hydrogen bonding. Nanocapsule formation is completed by free radical polymerization.

Each component of this nanocapsule is essential for cellular delivery. Inclusion of the imidazole-containing monomer provided a mechanism for endosomal/lysosomal escape. Without this, the sgRNA/Cas9 RNP would be destroyed before exerting its effects. A crosslinker containing a disulfide (–S-S–) bond produced a covalently linked, yet biodegradable, shell around the cargo. And finally, to reduce potential recognition by the immune system and increase the circulation half-life, a PEG outer shell was introduced. The PEG outer shell also provides a chemical handle for attaching fluorescent dyes or targeting ligands onto the RNP nanocapsule.

Engineered Probiotics as Systemic Therapeutic Delivery Platform

UW–Madison researchers have developed bacteria engineered to systemically deliver a therapeutic polypeptide into a subject without the bacteria being substantially introduced into the bloodstream. This platform could be used to non-invasively increase systemic levels of hormones, peptides and potentially single-chain antibodies.

Using this new approach, the researchers engineered a lactic acid bacteria, Lactobacillus reuteri, to systemically deliver interleukin-22 (IL-22) in mice. The method is not limited to IL-22; other potential polypeptides include IL-35, insulin, leptin, a peptide inhibitor of PCSK9 and endolysin.

Enhanced Drug Delivery Across the Blood-Brain Barrier: pH-Dependent Antibodies Targeting the Transferrin Receptor

UW–Madison researchers have developed several new single-chain antibody fragments to the transferrin receptor which exhibit increased dissociation at pH 5.5. Such targeting antibodies could have immense potential for drug delivery into and across target cells including cancer cells and the BBB.

Unlike other anti-TfR antibodies in development for cancer or brain delivery, the new antibodies have been endowed with pH-sensitivity resulting in differential trafficking and increased intracellular accumulation up to 2.6 times their wild-type parent.

Reagents for Bioreversible Protein Esterification

UW–Madison researchers have developed an optimized diazo compound, derived from phenylglycine amide, for converting carboxylate groups into an ester in high yield in buffered water. The ensuing esters are labile to esterase enzymes such as reside in all human cells, making the modification bioreversible. The novel compound is small, avoids deleterious side reactions and has a modularity that enables broad utility.

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, 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.

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.

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.

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.

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. 

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.

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.

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.

Phage-Cured Lactobacillus Strains for Therapeutic Delivery

Using a newly developed counterselection method and promoter construct, UW–Madison researchers have created a modified strain of L. reuteri cured of two prophages and variations thereof. In the process of deleting the phages from the genome the researchers also deleted the recognition site (attB), thereby preventing future phage integration at the site.

The approach used to develop the novel strain does not induce DNA damage and effectively eliminates bacteriophages that could otherwise reactivate and lyse the host cell. The strain is more robust for surviving transit through the gastrointestinal tract, which the researchers envision will enhance its ability to deliver therapeutic proteins in vivo.