Browsing by Subject "Conjugation"
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Item Albumin as a drug delivery carrier to overcome biological barriers in cancer(2022-10-06) Liu, Xinquan; Ghosh, Debadyuti; Smyth, Hugh D; Cui, Zhengrong; Brock, AmyIn disease, especially cancer, biological barriers impede the delivery of therapeutic agents to effectively reach their intended targets after administration. Developing delivery strategies that understand and take into account the biological environment is critical to improve the efficiency of drug delivery. Nature provides a biological toolkit from which effective drug delivery systems can be developed. In particular, albumin is an attractive delivery carrier with favorable intrinsic properties to overcome intracellular and extracellular delivery barriers in cancer. In a subset of cancers, the RAS family of oncogenes (KRAS, HRAS, NRAS) are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression, including hyperactive cell proliferation and metabolism. As a result, drug delivery targeting RAS-driven tumors has been a long-standing challenge in cancer therapy. Looking for solutions inspired by nature, recent observations indicate that extracellular nutrients, including glucose, lipids, and albumin, were found to be actively scavenged by mutant RAS activated cancer cells via macropinocytosis to fulfill the energetic requirements of cancer cells to survive and proliferate. Here, we exploit this mechanism to deliver albumin nanoparticles in cancer cells harboring activating KRAS mutations. We synthesized stable albumin nanoparticles that demonstrate significantly greater uptake in cancer cells with activating mutations of KRAS than monomeric albumin (i.e. dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these nanoparticles exhibit significantly increased uptake in mutant KRAS cancer cells than wild-type KRAS cells by macropinocytosis. Importantly, we demonstrate that their uptake is driven by KRAS. This nanoparticle-based strategy targeting KRAS-driven macropinocytosis is a facile approach towards improved delivery into KRAS-driven cancers. Through our studies with albumin nanoparticles, it was realized that albumin itself potentially possesses excellent pharmacokinetics that could be leveraged to improve drug delivery. To further harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drugs would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate, and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy. Collectively, these findings suggest that albumin can serve as an effective drug delivery carrier for drug delivery in solid tumors when the intrinsic properties of albumin were carefully utilized.Item Regulation of ATG5 and the ATG12–ATG5-ATG16L1 complex in prostate cancer(2015-05) Wible, Daric John; Wright, Casey Wyatt; Bratton, Shawn B.; Tang, Dean G; De Lozanne, Arturo; Fischer, Janice AAutophagy is a highly conserved pathway in which an autophagosome envelops cytoplasmic cargo and delivers it to the lysosome for degradation in order to maintain cellular homeostasis or survival in response to stress. The ATG12–ATG5-ATG16L1 complex functions as an essential regulator of autophagosome formation. We have discovered that DU145 prostate cancer (PCa) cells have a splice donor-site mutation that triggers aberrant splicing of ATG5 and leads to the proteasomal degradation of ATG12 and ATG16L1, thus completely inactivating autophagy. We demonstrate that ATG5, ATG12, and ATG16L1 are coordinately degraded when not associated with the complex and that the ATG5-ATG16L1 interaction is essential for preventing ubiquitination and turnover, thereby facilitating ATG12 conjugation. We also show that this interaction can be disrupted through alternative ATG5 splicing and by ATG5 genetic mutations that have been identified in human tumors. Meta-analysis of available mRNA expression data indicates that ATG5 is significantly downregulated in PCa. We confirmed previous reports that found prostate cancers have frequent deletions of the 6q21 locus containing ATG5. However, mRNA expression of neighboring genes is largely unaffected, indicating ATG5 can also be selectively downregulated though other mechanisms. Together, this suggests that ATG5 functions as a tumor suppressor gene that can be inactivated by a variety of different mechanisms. ATG5 is more significantly underexpressed than many established PCa tumor suppressor genes and is also underexpressed in PCa metastases compared to primary tumors. This implies that ATG5 is also a tumor suppressor in advanced PCa. ATG5 re-expression in ATG5-deficient DU145 PCa cells resulted in dramatic suppression of xenograft tumor growth, indicating that ATG5 is a functional PCa tumor suppressor gene. Therefore, autophagy may actually be tumor suppressive at both early and late stages of prostate tumorigenesis, which suggests that autophagy inhibition may be counterproductive for the treatment of advanced prostate cancers.Item Stimulus-responsive delivery systems for enabling the oral delivery of protein therapeutics exhibiting high isoelectric point(2015-05) Koetting, Michael Clinton; Peppas, Nicholas A., 1948-; Contreras, Lydia M; Ellison, Christopher J; Stachowiak, Jeanne C; Truskett, Thomas MProtein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption.Item Textured thin metal shells on metal oxide nanoparticles with strong NIR absorbance and high magnetization for imaging and therapy(2010-12) Ma, Li, doctor of chemical engineering; Feldman, Marc David; Johnston, Keith P., 1955-; Milner, Thomas E.; Sokolov, Konstantin V.; Mullins, C. B.; Hwang, Gyeong S.The ability of sub 100 nm nanoparticles to target and modulate the biology of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A general mechanism is presented for thin autocatalytic growth on nanoparticle substrates (TAGS), as demonstrated for a homologous series of < 5 nm textured Au coatings on < 42 nm iron oxide cluster cores. Very low Au supersaturation levels are utilized to prevent commonly encountered excessive autocatalytic growth that otherwise produce thick shells. The degree of separation of nucleation to form the seeds from growth is utilized to control the morphology and uniformity of the thin Au coatings. The thin and asymmetric Au shells produce strong near infrared (NIR) absorbance with a cross section of ~10⁻¹⁴ m², whereas the high magnetic content per particles provides strong r2 spin-spin magnetic relaxivity of 200 mM⁻¹s⁻¹. TAGS may be generalized to a wide variety of substrates and high energy coatings to form core-shell nanoparticles of interest in a variety of applications as diverse as catalysis and bionanotechnology. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast both in cell culture and an in vivo rabbit model of atherosclerosis. A novel conjugation technique further allows covalent binding of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (Ab) to the nanoclusters for highly selective targeting to EGFR over expressing cancer cells. AlexaFluor 488 tagged Ab nanocluster conjugates were prepared to correlate the number of conjugated Abs with the hydrodynamic diameter. The high targeting efficacy was evaluated by dark field reflectance imaging and atomic absorbance spectrometry (AAS). Colocalization of the nanoparticles by dual mode in-vitro imaging with dark field and fluorescence microscopy demonstrates the Abs remained attached to the Au surfaces. The extremely high curvature of the Au shells with features below 5 nm influence the spacing and orientations of the Abs on the surface, which has the potential to have a marked effect on biological pathways within cells. These targeted small multifunctional nanoclusters may solve some key molecular imaging challenges for cancer and atherosclerosis.