Dendrimer Conjugation Enhances Tumor Penetration and Cell Kill of Doxorubicin in 3D Coculture Lung Cancer Models

Background: Doxorubicin (DOX) is a potent chemotherapeutic widely used for solid tumors (1). Despite high efficacy in 2D cell culture, DOX efficacy does not translate to in vivo lung cancer models (2). Major side effects such as cardiotoxicity may be alleviated with nano-based drug delivery systems (nanoDDS). However, tumor penetration of DOX and DOX-nanoDDS is largely unknown and is an additional barrier to effective clinical therapy (3). Here we describe a nanoDDS capable of enhancing the penetration of DOX. Methods: DOX was conjugated to generation 4 poly(amido-amine) dendrimers through (GFLG) tumor- liable bond. G4SA-GFLG-DOX was synthesized/characterized. spheroids were formed of (A549) lung adenocarcinoma cells and (3T3) fibroblasts. Spheroids were characterized for ECM components with immunohistochemistry. Confocal microscopy was used to evaluate the penetration, internalization, and colocalization of DOX and G4SA-GFLG-DOX. MTT assay and Caspase 3/7 to assess 2D and 3D cytotoxicity. Flow cytometry to determine cells uptake. Results: DOX conjugation to dendrimer resulted in G4SA-GFLG-DOX with ~5.5 DOX, 10±1 nm hydrodynamic diameter, and a -17±3 mV zeta-potential. Spheroids of (A549:3T3) were ECM- rich, developed ECM containing collagen-I, hyaluronan, laminin, and fibronectin. While DOX and G4SA-GFLG-DOX had similar toxicities in 2D model, G4SA-GFLG-DOX demonstrated a 3.1-fold greater penetration into spheroids compared to DOX and correlated to a greater efficacy as measured by caspase 3/7 activity. Also, flow cytometry showed higher uptake of G4SA- GFLG-DOX in cancer cells compared to fibroblasts. Conclusion: The work demonstrates enhanced penetration of DOX, via dendrimer conjugation, into an ECM- rich 3D lung cancer model. The enhanced penetration of G4SA-GFLG-DOX correlated with greater antitumor efficacy. Acknowledgements: We acknowledge partial financial support from the Center for Pharmaceutical Engineering and Sciences - School of Pharmacy at VCU. This study was supported by VCU Quest for Distinction and NSF (DRM #1508363). Microscopy was performed at the VCU Microscopy Facility, supported, in part, by funding from NIH-NCI Cancer Center Support Grant P30 CA016059. RA would like to acknowledge King Faisal University (KFU) and Saudi Arabian Cultural Mission (SACM) for a scholarship.https://scholarscompass.vcu.edu/gradposters/1091/thumbnail.jp

Doxorubicin-Loaded Human Serum Albumin Submicron Particles: Preparation, Characterization and In Vitro Cellular Uptake

Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation-Crosslinking-Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (-13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase®) within the same time. In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy

Recombinant methioninase combined with doxorubicin (DOX) regresses a DOX-resistant synovial sarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

Synovial sarcoma (SS) is a recalcitrant subgroup of soft tissue sarcoma (STS). A tumor from a patient with high grade SS from a lower extremity was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) mouse model. The PDOX mice were randomized into the following groups when tumor volume reached approximately 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks; G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G4 DOX (3mg/kg), i.p. weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks). On day 14 after treatment initiation, all therapies significantly inhibited tumor growth compared to untreated control, except DOX: (DOX: p = 0.48; rMETase: p < 0.005; DOX combined with rMETase < 0.0001). DOX combined with rMETase was significantly more effective than both DOX alone (p < 0.001) and rMETase alone (p < 0.05). The relative body weight on day 14 compared with day 0 did not significantly differ between any treatment group or untreated control. The results indicate that r-METase can overcome DOX-resistance in this recalcitrant disease

Functional Blockade of E-Selectin in Tumor-Associated Vessels Enhances Anti-Tumor Effect of Doxorubicin in Breast Cancer

Chemotherapy is a mainstay of treatment for solid tumors. However, little is known about how therapy-induced immune cell infiltration may affect therapy response. We found substantial CD45+ immune cell density adjacent to E-selectin expressing inflamed vessels in doxorubicin (DOX)-treated residual human breast tumors. While CD45 level was significantly elevated in DOX-treated wildtype mice, it remained unchanged in DOX-treated tumors from E-selectin null mice. Similarly, intravenous administration of anti-E-selectin aptamer (ESTA) resulted in a significant reduction in CD45+ immune cell density in DOX-treated residual tumors, which coincided with a delay in tumor growth and lung metastasis in MMTV-pyMT mice. Additionally, both tumor infiltrating T-lymphocytes and tumor associated-macrophages were skewed towards TH2 in DOX-treated residual breast tumors; however, ESTA suppressed these changes. This study suggests that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift

Morphometric study of myocardial changes during doxorubicin-induced cardiomyopathy in mice

Doxorubicin (DOX) is one of the most effective anti-cancer drugs in oncology, but may cause a cumulative dose-dependent cardiomyopathy in a number of cancer patients. The effect of DOX on the heart was studied in mice treated with i.v. injections of 2 mg/kg by measuring morphometric parameters, including nuclear index (number of non-myocytes/number of myocyte nuclei), reticulin index (reticulin area/number of myocyte transsections), nuclear transsectional area, myocyte transsectional area, capillary index (number of capillaries/number of myocyte transsections) and capillary transsectional area. The highest significant difference between control mice and DOX-treated mice was observed immediately after the 12th dose of DOX except for the two capillary parameters. The highest level of significance for these two parameters was obtained 12 weeks after the end of DOX treatment. In contrast to the observations in rats, mice did not develop a nephrotic syndrome during treatment with DOX. The morphometric analysis of myocardial changes in mice, as a quantitative and objective method, seems to be a good model for comparative studies on cardiomyopathy induced by anthracycline analogues

The disintegrin echistatin in combination with doxorubicin targets high-metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models.

Echistatin, a cyclic RGD peptide, which is an antagonist of αvβ3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing αvβ3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by echistatin (P<0.05) as was overall growth. A doxorubicin (DOX)-echistatin combination inhibited orthotopic tumor growth compared to untreated control (P<0.01) or DOX alone (P<0.05) in nude mice. Tumor-bearing mice treated with the DOX-echistatin combination survived longer than those treated with DOX alone or control PBS (P<0.01 and P<0.01, respectively). Echistatin also inhibited experimental lung metastasis of 143B-LM4 cells in nude mice. These results suggest that DOX in combination with a disintegrin has potential to treat osteosarcoma and that αvβ3 integrin may be a target for osteosarcoma

And All Was Cold As Any Stone: Death and the Critique of Representation

And All Was Cold As Any Stone: Death and the Critique of Representatio

Logic and Performance: Translating the Poetics into Medieval Scholasticism

Logic and Performance: Translating the Poetics into Medieval Scholasticis

Drug-Loaded, Bivalent-Bottle-Brush Polymers by Graft-through ROMP

Graft through ring-opening metathesis polymerization (ROMP) using ruthenium N heterocyclic carbene catalysts has enabled the synthesis of bottle-brush polymers with unprecedented ease and control Here we report the first bivalent-brush polymers, these materials were prepared by graft through ROMP of drug-loaded poly(ethylene glycol) (PEG) based macromonomers (MMs) Anticancer drugs doxorubicin (DOX) and camptothecin (CT) were attached to a norbornene alkyne-PEG MM via a photocleavable linker ROMP of either or both drug loaded MMs generated brush homo and copolymers with low polydispersities and defined molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light All of the CT and DOX polymers were at least 10 fold more toxic to human cancer cells after photoinitiated drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irradiation Graft through ROMP of drug loaded macromonomers provides a general method for the systematic study of structure function relationships for stimuli responsive polymers in biological systems

The Behavior of Benzidine toward Selenic and Telluric Acids

Within quite recent years, benzidine (p-diaminodiphenyl) has come into use as a precipitant for the sulfate ion. It was first applied as a quantitative reagent for the determination of sulfate by Raschig in 1903. Other investigators subsequently introduced modifications in the original method of Raschig and succeeded in obtaining very satisfactory analyses with this reagent. For example, in the analysis of water samples which contain iron salts, hydroxylamine hydrochloride is added to prevent oxidation of the benzidine. The precipitated benzidine sulfate is collected in the usual way and is either weighed direct or titrated with sodium hydroxide, using phenolphthalein as an indicator. Titration is rendered possible by the very weak basic properties of benzidine. Bruckmiller states that the benzidine method for sulfates in water compares favorably with the time-honored barium chloride method in point of accuracy, and has the advantage of being more rapid