News Column

Researchers Submit Patent Application, "Enzyme Directed Assembly of Particle Theranostics", for Approval

July 28, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Gianneschi, Nathan C. (San Diego, CA); Hahn, Michael (San Diego, CA); Mattrey, Robert (San Diego, CA), filed on June 11, 2012, was made available online on July 17, 2014 (see also The Regents Of The University Of California).

The patent's assignee is The Regents Of The University Of California.

News editors obtained the following quote from the background information supplied by the inventors: "There is an ever-increasing knowledge base concerning the molecular signatures of specific diseases and their potential in personalized medicine; however, the translation of this information into clinical practice lags significantly behind. 'Theranostic' agents are of particular interest since they combine in vivo imaging for diagnostics and therapeutics within a single system.

"There is a tremendous need for novel and effective approaches to molecular imaging in vivo, since current structural imaging techniques do not capitalize on the molecular basis of disease to add specificity. While structure imaging is oftentimes sufficient to answer general clinical questions, it has been inadequate in assessing molecular characteristics of diseased tissues (i.e., tumors). At times, structural imaging techniques are unable to discern benign from malignant tissue, such as lymph nodes or lung nodules. The methods and compositions described herein can fill the void and thus expand the reach of therapy by allowing the visualization, characterization, and measurement of biological processes at the molecular and cellular levels."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "Described herein are programmable stimuli responsive nanomaterials for detecting and treating disease. Further provided is an in vivo assembly of nanoscale objects of specific size, shape, photophysical, magnetic, and pharmacokinetic properties in response to disease-associated enzymatic signals. This approach has been termed Enzyme-directed Assembly of Particle Theranostics (EDAPT). Described herein are methods of making and using DNA-programmable and peptide-programmable materials capable of accumulating and subsequently activating in diseased tissue while evading non-specific accumulation. The present disclosure will alleviate problems limiting the efficacy of current delivery strategies for both diagnostics and therapeutics. Provided herein is a novel diagnostic and therapeutic system directed at diseased tissues.

"Aberrantly high activity of pro-oncogenic enzymes is one significant hallmark among the multitude of changes present in tumors. Many such overactive enzymatic biomarkers have been identified but using their presence in diagnostic or therapeutic strategies has lagged behind their discovery. One particularly promising enzyme family, the matrix metalloproteinases (MMPs), has been extensively studied in all phases of cancer progression. These enzymes contribute to oncogenesis and metastasis through several mechanisms and their activity has been observed to increase dramatically as the tumor becomes more aggressive. Increased MMP expression has also been shown to correlate significantly with prognosis in a wide range of malignancies. Described herein is use of molecular imaging of MMP activity as a potential diagnostic, prognostic, and treatment stratification tool. Molecular imaging of MMP activity can be utilized in a similar fashion as FDG-PET, currently used for non-invasive monitoring of tumor molecular activity in patients. The presently disclosed methods and compositions provide clinicians a new means to determine if a given treatment regimen is active at early time points in each specific patient, thus facilitating personalized medicine. Provided herein is a novel strategy for the molecular imaging of MMP activity in vivo that can enable specific and sensitive MMP detection in various cancers.

"Provided herein is a method of detecting a diseased tissue in a subject comprising: (a) obtaining a hydrophilic polymer probe comprising: a hydrophilic polymer containing an enzyme cleavable moiety, and a visualizable label; (b) administering a hydrophilic polymer probe to the tissue in an amount sufficient to provide a detectable image; allowing cleavage of a hydrophilic polymer probe by an enzyme, wherein the cleaved hydrophilic polymer probe self-assembles into an amphiphilic polymer aggregate, comprising of amphiphilic polymer and a visualizable label, at the cellular location to be imaged; and (d) imaging the tissue, wherein a detectable image of the self-assembled particle aggregate in the tissue indicates the presence of a diseased tissue. The enzyme cleavable moiety is a peptide-based structure or a DNA-based structure. The visualizable label is a fluorophore, quencher, Gd.sup.3+ reporter or combinations thereof.

"Provided herein is a method of detecting a cancerous tissue in a subject comprising: (a) obtaining a hydrophilic polymer probe comprising: a hydrophilic polymer containing MMP-cleavable polypeptide, and a visualizable label; (b) administering a hydrophilic polymer probe to the tissue in an amount sufficient to provide a detectable image; allowing cleavage of a hydrophilic polymer probe by a cancer-associated enzyme, wherein the cleaved hydrophilic polymer probe self-assembles into an amphiphilic polymer aggregate, comprising of amphiphilic polymer and a visualizable label, at the cellular location to be imaged; and (d) imaging the tissue, wherein a detectable image of the self-assembled particle aggregate in the tissue indicates the presence of a cancerous tissue. The cancer-associated enzyme is a nuclease (endonuclease, exonuclease), a protease, or a matrix metalloprotease (MMP-2, MMP-9).

"Provided herein is a method of treating a subject having cancer, the method comprising: (a) obtaining a hydrophilic polymer probe comprising: a hydrophilic polymer containing an enzyme cleavable moiety, targeting structure, and a therapeutic agent; (b) administering a hydrophilic polymer probe to the tissue in an amount sufficient to provide a therapeutic dosage to the subject; allowing the localization of a hydrophilic polymer probe to the targeted tissue in the subject as directed by the targeting polypeptide; and (d) allowing cleavage of a hydrophilic polymer probe by an enzyme, wherein the cleaved hydrophilic polymer probe self-assembles into an amphiphilic polymer aggregate, comprising of amphiphilic polymer and a therapeutic agent, at the cellular location to be treated, thereby treating a subject with cancer. The enzyme cleavable moiety is a peptide-based structure or a DNA-based structure.

"Provided herein is a method of treating a subject having cancer, the method comprising: (a) obtaining a hydrophilic polymer probe comprising: a hydrophilic polymer containing an MMP-cleavable polypeptide, targeting structure, and a therapeutic agent; (b) administering a hydrophilic polymer probe to the tissue in an amount sufficient to provide a therapeutic dosage to the subject; allowing the localization of a hydrophilic polymer probe to the targeted tissue in the subject as directed by the targeting polypeptide; and (d) allowing cleavage of a hydrophilic polymer probe by an enzyme, wherein the cleaved hydrophilic polymer probe self-assembles into an amphiphilic polymer aggregate, comprising of amphiphilic polymer and a therapeutic agent, at the cellular location to be treated, thereby treating a subject with cancer. The targeting structure is a peptide-based structure or a nucleic acid-based structure.

"Provided herein is an enzymatically-directed self-assembled amphiphilic polymer aggregate comprising: an amphiphilic polymer, which comprises of a hydrophilic cryptic-amphiphile with a cleaved MMP-specific polypeptide, and a labeling group.

"Provided herein is an enzymatically-directed self-assembled amphiphilic polymer aggregate comprising: an amphiphilic polymer, which comprises of a hydrophilic cryptic-amphiphile with a cleaved nucleic acid structure, and a labeling group.

"Provided herein is a hydrophilic polymer probe, which is prepared by a preparation method comprising synthesizing a polymer using the method of ring-opening metathesis polymerization, and incorporating labeling groups, therapeutic agents and/or targeting groups into said synthesized polymer.

"Provided herein is a method of treating a diseased tissue of a subject's body, comprising providing a therapeutic agent conjugated to hydrophophilic polymer probe in a manner to direct self-assembly of the hydrophilic polymer into amphiphilic polymer aggregates and delivery of the drug to the diseased tissue.

"Provided herein is a method of treating a MMP-mediated cancerous tissue of a subject's body, comprising providing a therapeutic agent conjugated to hydrophophilic polymer probe in a manner to direct self-assembly of the hydrophilic polymer into amphiphilic polymer aggregates and delivery of the drug to the MMP-mediated cancerous tissue. The MMP-mediated cancerous tissue is a MMP-mediated malignancy, sarcoma, or metastasis.

"Presented herein is a method of imaging a diseased tissue of a subject's body, comprising providing a labeling agent conjugated to hydrophophilic polymer probe in a manner to direct self-assembly of the hydrophilic polymer into amphiphilic polymer aggregates and delivery of the labeling agent to the diseased tissue.

"Presented herein is a method of imaging a MMP-mediated cancerous tissue of a subject's body, comprising providing a labeling agent conjugated to hydrophophilic polymer probe in a manner to direct self-assembly of the hydrophilic polymer into amphiphilic polymer aggregates and delivery of the labeling agent to the MMP-mediated cancerous tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 shows enzyme-directed assembly of particle theranostics. FIG. 1A shows the formation of self-assembled nanoparticles from peptide-polymer amphiphile following cleavage by the enzyme MMP. FIG. 1B shows the use of self-assembled nanoparticles to target tumor tissue in the body.

"FIG. 2 illustrates the pharmocokinetic profile and detection properties of an enzyme-responsive micellar nanomaterial

"FIG. 3 illustrates cryptic-amphiphile structures.

"FIG. 4 shows formation of a Type-2 cryptic-amphiphile.

"FIG. 5 illustrates nanoparticle self-aggregation in response to enzyme cleavage of peptide substrates.

"FIG. 6 shows a schematic diagram of dye labeled self-assembled aggregates. Nanoparticles were designed to generate a unique FRET signal with fluorescein-polymers and rhodamine-polymers.

"FIG. 7 shows the FRET signal at an appropriate concentration for the synthesis reaction.

"FIG. 8 shows a study to determine the optimal micelle concentration for a FRET based nanoparticle. A concentration of the amphiphile is needed to create particle to unimer equilibrium.

"FIG. 9 shows that MRI-agent chelates for direct incorporation into polymer backbone.

"FIG. 10 shows multienzymatic responsive micellular nanoparticles.

"FIG. 11 shows phosphorylation and dephosphorylation in reversible cycling of particle morphology. The top panel shows that the phosphorylated substrate adopts an aggregate morphology (left) and the dephosphorylated substrate does not (right). Phosphorylation of the substrate is induced by PKA. The bottom panel shows proteolysis at the particle shell. Proteolysis of the MMP substrate is controlled by MMP2. M2 micelles form aggregates in the presence of MMP2 while M1 micelles do not.

"FIG. 12 illustrates an assembly of peptide-polymer amphiphiles (PPAs) to generate fluorogenic micellar nanoparticles. Polymers are labeled with peptides and dyes, post-polymerization with block sizes determined by SEC-MALS analysis and spectroscopy. Degree of dye incorporation (m), was between 1 and 2 for both PPA-1 and PPA-2.

"FIG. 13 illustrates TEM, DLS and fluorescence spectroscopy of fluorogenic micelles. FIG. 13A shows TEM of 30 nm M3. FIG. 13B shows DLS of M1, M2 and M3 showing hydrodynamic diameters (Dh) in the range of 30-40 nm. FIG. 13C shows fluorescence emission spectra of M1, M2 and FRET-micelle, M3 upon excitation at 470 nm. FIG. 13D shows the ratio of normalized emission intensity for maxima at 563 nm (rhodamine) and 512 nm (fluorescein) over a range of concentrations of PPA-1 and PPA-2 upon excitation at 470 nm. Arrow indicates onset of detectable FRET.

"FIG. 14 illustrates the time-domain fluorescence intensity decay analysis of M1 and M3 for determination of structural parameters. FIG. 14A shows the fluorescence lifetime of M3 and M1 fit to a distance distribution function, and single exponential respectively (red lines) giving tD=3.98+/-0.01 ns (from M1 data) for unquenched fluorescein. FIG. 14B shows for M3, a range of distances between donor and acceptor (D and A) is considered, expressed as a probability function P(r). The mean distance, 3.6 nm, can in turn be used to determine tDA=0.29 ns. Radius of the micelles was determined by TEM (FIG. 13).

"FIG. 15 illustrates the response of mixtures of M1 and M2 to MMPs. FIG. 15A shows the fluorescence spectra of M1 and M2 (0.5 .mu.M each with respect to PPA) with and without MMP-9 (10 nM) at times indicated following enzyme addition; .lamda.ex=470. FIG. 15B-D shows fluorescence intensity vs time plots via plate reader analysis, to monitor rearrangement of PPA-1 and PPA-2 into new FRET active aggregates; .lamda.ex=490 and .lamda.em=590 nm. FIG. 21B shows detection of MMP-9 down to 10 pM of enzyme with M1 and M2 (at 0.5 .mu.M, [PPA]). FIG. 15C shows detection of MMP-9 at 10 nM with varying concentrations of M1 and M2 shown with respect to [PPA], detectable down to 20 nM of polymer. FIG. 15D shows detection of cell-secreted (WPE1-NA45 cells) MMP-2 and -9 with varying concentrations of M1 and M2 shown with respect to [PPA]. Cells were seeded at 1.6.times.10.sup.4 cells/well in a clear bottom 96-well plate in DMEM. After 24 hrs, cell medium was added to solutions of M1 and M2. MMP-2 and -9 were at 0.048 nM and 0.005 nM respectively as quantified by an ELISA assay. Control was the non-MMP expressing MCF-7 cell-line cultured in the same manner. All reactions run in PBS, unless otherwise noted. FIG. 15E-F shows TEM of M1 and M2 before, and after 24 hrs following MMP-9 treatment.

"FIG. 16 depicts a table of detection of MMP-9 at 10 mM in blood serum doped DMEM, a cell growth medium Detection of MMP in blood serum doped DMEM media via significant shortening in fluorescence lifetime indicating particle fusion upon cleavage of PPAs within M1 and M2.

"FIG. 17A illustrates monomer synthesis of tert-butyl-(2-((2S)-bicyclo[2.2.1]hept-5-ene-2-carboxamido)ethyl)carbamat- e. FIG. 17B illustrates polymer synthesis.

"FIG. 18 illustrates a SEC-MALS intensity plot of initially prepared 1.sub.21-b-2.sub.6-b-3.sub.3 (blue) and following conjugation with Peptide 1 (green). SEC-MALS: 1.sub.21-b-2.sub.6-b-3.sub.3; Mn=7459 g/mol, PDI=1.053. Peptide conjugate of 1.sub.21-b-2.sub.6-b-3.sub.3; Mn=15270 g/mol, PDI=1.164.

"FIG. 19 depicts a table of polymers, PPAs and resulting micelles.

"FIG. 20A shows a graph of product vs time for reactions of M4 with MMP-9 at various substrate concentrations. FIG. 20B shows a graph of Initial rate vs substrate concentration. a indicates the literature value of Kcat/KM for standard peptide with MMP-9.

"FIG. 21A illustrates MMP-9 cleavage of M1 (green trace) and Peptide-1 (blue trace). Red trace is intact Peptide-1 without treatment with MMP-9. FIG. 21B illustrates a MALDI-TOF mass spectrum. MALDI-MS of Peptide-1 fragment (left) and fragment from M1 (right) cleaved by MMP-9. Mass calcd: 1398.6, Obs: 1399.7 (A) and 1399.2 (B).

"FIG. 22 shows micelle counting via 20 nm Au NPs calibration visualized by TEM. 20 .mu.L of M3 was mixed with 20 .mu.L of 20 nm Au NPs at concentration of 7.times.10.sup.14 particles/L. 1243 M3 and 158 Au NPs were counted. TEM images shown here are representative of M3 mixed with Au NPs. M3 was counted as 5.51.times.10.sup.15 particles/L after calibration by Au NPs. Arrows indicate some representative 20 nm Au NPs visible clearly from TEM images as solid spheres, as opposed to open circles for the organic matter stained by uranyl acetate.

"FIG. 23 shows a table of the weight average molar mass and aggregation number of M1, M2 and M3 from SLS. FIG. 23 also shows a graph of the volume % vs the hydrodynamic diameter of the aggregates. It illustrates DLS of M1/M2 micelle mixtures mixed with non-activated MMP-9 (black; left) or activated MMP-9 (red; right).

"FIG. 24 shows DLS (top) and TEM (bottom) data of M3 mixed with MMP-9.

"FIG. 25 shows results of PPA injections into nude mice expressing HT1080 tumors. FIG. 25A shows a diagram of PPA-mediated aggregate formation. FIG. 25B shows the expression of the nanoparticles in the mice. FIG. 25C shows fluorescence of the nanoparticles in tumors.

"FIG. 26 shows embodiments of a DNA-programmed micelle design. A copolymer 1.sub.38-b-2.sub.18 is conjugated with a DNA moiety, such as DNA-1, DNA-2 and DNA-3. The DNA-polymer can include other moieties such as PEG or F. The DNA-polymer undergoes cleavage and forms a micelle. The micelle comprises a DNA shell and a phenyl core.

"FIG. 27 shows analysis of the DNA-programmed micelles. The top panel shows SLS analysis and AFM analysis. The bottom panel shows a TEM image of the micelles.

"FIG. 28 shows another example of DNA-programmed nanomaterial. FIG. 28A shows the DNA sequences. FIG. 28B shows the reaction used to form micelles and aggregates with DNA.sub.s based nanomaterial. FIG. 28C shows images of the micelles (left) and aggregates (right).

"FIG. 29 shows another example of DNA-programmed nanomaterial.

"FIG. 30 illustrates that programmed amphiphilicity of the synthetic polymeric nanoparticles can enable reversible morphology changes. Altering the hydrophilic DNA-brush and/or hydrophobic particle core can allow a nanoparticle to change from a sphere to a cylinder (e.g., fiber) upon DNA cleavage by a nuclease (i). A nanoparticle that is a cylinder can change shape to be a sphere upon DNA annealing (ii) and change to the hydrophilic DNA-brush.

"FIG. 31 illustrates an increase in fluorescence of fluorescent DNA-polymer micelles (substrate) in the presence of enzyme (DNAzyme).

"FIG. 32 illustrates fluorescence in the DNA-polymer nanoparticles during morphological changes induces by DNAzymes and the DNA sequence in the particle shell.

"FIG. 33 shows that the length of the nano fibers can be controlled by the nanomaterial.

"FIG. 34 shows a diagram of switching the morphology of polymeric nanoparticles.

"FIG. 35 shows the conformational differences between spherical and cylindrical nanoparticles. FIG. 35F shows a schematic diagram of the DNA-polymer nanomaterial. Using J77 murine macrophage cells, we add either 0.1 nmole rhodamine and fluorescein co-labeled DNA-polymer nanofibers (FIG. 35A-D) or nanospheres (FIG. 35E) to the cells. Then, we added 0.1 nmole of complementary DNA from another 1 hr (FIG. 35B, 2 hrs (FIG. 35C) and 4 hrs (FIG. 35D).

"FIG. 36 shows fluorescence detected in mice injected with rhodamine and fluorescein labeled spherical and fiber nanomaterial structures. FIG. 36A shows fluorescence of the fibers. FIG. 36B compares the fluorescence of the spheres alone, the fibrils alone and the fibrils incubated with complementary DNA.

"FIG. 37A shows the fluorescence detected in organs from mice injected with rhodamine and fluorescein labeled spherical and fiber nanomaterial structures. FIG. 37B shows the fibers present in serum 24 hours post-injection."

For additional information on this patent application, see: Gianneschi, Nathan C.; Hahn, Michael; Mattrey, Robert. Enzyme Directed Assembly of Particle Theranostics. Filed June 11, 2012 and posted July 17, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2844&p=57&f=G&l=50&d=PG01&S1=20140710.PD.&OS=PD/20140710&RS=PD/20140710

Keywords for this news article include: Cancer Gene Therapy, DNA Research, Emerging Technologies, Enzymes and Coenzymes, Fluoresceins, Hydrocarbons, Molecular Imaging, Nanoparticle, Nanotechnology, Oncology, Peptides, Personalized Medicine, Proteins, Proteomics, Spiro Compounds, The Regents Of The University Of California.

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Source: Cancer Gene Therapy Week


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