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1.  Synthetic Biology for Therapeutic Applications 
Molecular pharmaceutics  2014;12(2):322-331.
Synthetic biology is a relatively new field with the key aim of designing and constructing biological systems with novel functionalities. Today, synthetic biology devices are making their first steps in contributing new solutions to a number of biomedical challenges, such as emerging bacterial antibiotic resistance and cancer therapy. This review discusses some synthetic biology approaches and applications that were recently used in disease mechanism investigation and disease modeling, drug discovery and production, as well as vaccine development, treatment of infectious diseases, cancer, and metabolic disorders.
PMCID: PMC4319687  PMID: 25098838
synthetic biology; disease mechanism; drug discovery; vaccine development; therapeutic treatment; cancer treatment; infectious diseases; metabolic disorders
2.  Targeted Nanogel Conjugate for Improved Stability and Cellular Permeability of Curcumin: Synthesis, Pharmacokinetics, and Tumor Growth Inhibition 
Molecular Pharmaceutics  2014;11(9):3112-3122.
Curcumin (CUR) is a unique natural compound with promising anticancer and anti-inflammatory activities. However, the therapeutic efficacy of curcumin was challenged in clinical trials, mostly due to its low bioavailability, rapid metabolism, and elimination. We designed a nanodrug form of curcumin, which makes it stable and substantially enhances cellular permeability and anticancer activity at standard oral administration. Curcumin was conjugated as an ester to cholesteryl-hyaluronic acid (CHA) nanogel that is capable of targeted delivery to CD44-expressing drug-resistant cancer cells. CHA-CUR nanogels demonstrated excellent solubility and sustained drug release in physiological conditions. It induced apoptosis in cancer cells, suppressing the expression of NF-κB, TNF-α, and COX-2 cellular targets similar to free curcumin. Pharmacokinetic/pharmacodynamic (PK/PD) studies also revealed improved circulation parameters of CHA-CUR at oral, i.p. and i.v. administration routes. CHA-CUR showed targeted tumor accumulation and effective tumor growth inhibition in human pancreatic adenocarcinoma MiaPaCa-2 and aggressive orthotropic murine mammary carcinoma 4T1 animal models. CHA-CUR treatment was well-tolerated and resulted in up to 13-fold tumor suppression, making this nanodrug a potential candidate for cancer prevention and therapeutic treatment.
PMCID: PMC4151794  PMID: 25072100
Curcumin; nanogel−drug conjugate; NF-κB inhibition; PK/PD parameters; animal cancer models
3.  Evaluation of 89Zr-pertuzumab in Breast Cancer Xenografts 
Molecular Pharmaceutics  2014;11(11):3988-3995.
Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2–specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with 89Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of 89Zr-pertuzumab in HER2–expressing BT-474 and HER2–nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2–expressing cells. In vivo evaluation of 89Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. 89Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of 89Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that 89Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.
PMCID: PMC4224522  PMID: 25058168
89Zr; pertuzumab; HER2; PET; trastuzumab; breast cancer xenograft; NOG
4.  44Sc: An Attractive Isotope for Peptide-Based PET Imaging 
Molecular Pharmaceutics  2014;11(8):2954-2961.
The overexpression of integrin αvβ3 has been linked to tumor aggressiveness and metastasis in several cancer types. Because of its high affinity, peptides containing the arginine–glycine–aspartic acid (RGD) motif have been proven valuable vectors for noninvasive imaging of integrin αvβ3 expression and for targeted radionuclide therapy. In this study, we aim to develop a 44Sc-labeled RGD-based peptide for in vivo positron emission tomography (PET) imaging of integrin αvβ3 expression in a preclinical cancer model. High quality 44Sc (t1/2, 3.97 h; β+ branching ratio, 94.3%) was produced inexpensively in a cyclotron, via proton irradiation of natural Ca metal targets, and separated by extraction chromatography. A dimeric cyclic-RGD peptide, (cRGD)2, was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with 44Sc in high yield (>90%) and specific activity (7.4 MBq/nmol). Serial PET imaging of mice bearing U87MG tumor xenografts showed elevated 44Sc-DOTA-(cRGD)2 uptake in the tumor tissue of 3.93 ± 1.19, 3.07 ± 1.17, and 3.00 ± 1.25 %ID/g at 0.5, 2, and 4 h postinjection, respectively (n = 3), which were validated by ex vivo biodistribution experiments. The integrin αvβ3 specificity of the tracer was corroborated, both in vitro and in vivo, by competitive cell binding and receptor blocking assays. These results parallel previously reported studies showing similar tumor targeting and pharmacokinetic profiles for dimeric cRGD peptides labeled with 64Cu or 68Ga. Our findings, together with the advantageous radionuclidic properties of 44Sc, capitalize on the relevance of this isotope as an attractive alternative isotope to more established radiometals for small molecule-based PET imaging, and as imaging surrogate of 47Sc in theranostic applications.
PMCID: PMC4128785  PMID: 25054618
scandium-44 (44Sc); arginine−glycine−aspartic acid (RGD) peptides; RGD dimer; integrin αvβ3; tumor angiogenesis; positron emission tomography (PET); molecular imaging
5.  PET Imaging of Fatty Acid Amide Hydrolase with [18F]DOPP in Nonhuman Primates 
Molecular Pharmaceutics  2014;11(11):3832-3838.
Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [11C]CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However, no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [18F]DOPP ([18F]3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate) has been identified as a promising 18F-labeled analogue based on rodent studies. The goal of this work is to evaluate [18F]DOPP in nonhuman primates to support its clinical translation. High specific activity [18F]DOPP (5–6 Ci·μmol–1) was administered intravenously (iv) to three baboons (2M/1F, 3–4 years old). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 μg/kg iv, 10 min prior to [18F]DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ∼95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [18F]DOPP covalently links to the active site of FAAH. Consistent with previous findings for [11C]CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter λk3 was therefore used to evaluate whole brain (WB) and regional binding of [18F]DOPP. Pretreatment studies showed inhibition of λk3 across all brain regions (WB baseline: 0.112 mL/cm3/min; 300 μg/kg URB597: 0.058 mL/cm3/min), suggesting that [18F]DOPP binding is specific for FAAH, consistent with previous rodent data.
PMCID: PMC4224570  PMID: 25004399
[18F]DOPP; fatty acid amide hydrolase; FAAH; positron emission tomography; PET; kinetic modeling
6.  Validating anti-metastatic effects of natural products in an engineered microfluidic platform mimicking tumor microenvironment 
Molecular pharmaceutics  2014;11(7):2022-2029.
Development of new, anti-metastatic drugs from natural products has been substantially constrained by the lack of a reliable in vitro screening system. Such a system should ideally mimic the native, three-dimensional (3D) tumor microenvironment involving different cell types and allow quantitative analysis of cell behavior critical for metastasis. These requirements are largely unmet in the current model systems, leading to poor predictability of the in vitro collected data for in vivo trials, as well as prevailing inconsistency among different in vitro tests. In the present study, we report application of a 3D, microfluidic device for validation of the anti-metastatic effects of twelve natural compounds. This system supports co-culture of endothelial and cancer cells in their native 3D morphology as in the tumor microenvironment, and provides real-time monitoring of the cells treated with each compound. We found that three compounds, namely sanguinarine, nitidine, and resveratrol, exhibited significant anti-metastatic or anti-angiogenic effects. Each compound was further examined for its respective activity with separate conventional biological assays, and the outcomes were in agreement with the findings collected from the microfluidic system. In summary, we recommend use of this biomimetic model system as a new engineering tool for high-throughput evaluation of more diverse natural compounds with varying anti-cancer potentials.
PMCID: PMC4310574  PMID: 24533867
microfluidics; metastasis; natural products; angiogenesis; cell co-culture
7.  PEG-Farnesyl Thiosalicylic Acid Telodendrimer Micelles as an Improved Formulation for Targeted Delivery of Paclitaxel 
Molecular Pharmaceutics  2014;11(8):2807-2814.
We have recently designed and developed a dual-functional drug carrier that is based on poly(ethylene glycol) (PEG)-derivatized farnesylthiosalicylate (FTS, a nontoxic Ras antagonist). PEG5K-FTS2 readily form micelles (20–30 nm) and hydrophobic drugs such as paclitaxel (PTX) could be effectively loaded into these micelles. PTX formulated in PEG5K-FTS2 micelles showed an antitumor activity that was more efficacious than Taxol in a syngeneic mouse model of breast cancer (4T1.2). In order to further improve our PEG-FTS micellar system, four PEG-FTS conjugates were developed that vary in the molecular weight of PEG (PEG2K vs PEG5K) and the molar ratio of PEG/FTS (1/2 vs 1/4) in the conjugates. These conjugates were characterized including CMC, drug loading capacity, stability, and their efficacy in delivery of anticancer drug PTX to tumor cells in vitro and in vivo. Our data showed that the conjugates with four FTS molecules were more effective than the conjugates with two molecules of FTS and that FTS conjugates with PEG5K were more effective than the counterparts with PEG2K in forming stable mixed micelles. PTX formulated in PEG5K-FTS4 micelles was the most effective formulation in inhibiting the tumor growth in vivo.
PMCID: PMC4123940  PMID: 24987803
paclitaxel; farnesyl thiosalicylic acid; dual function; nanomicelles; targeted delivery
8.  Evaluation of 99mTc-Probestin SPECT As a Novel Technique for Noninvasive Imaging of Kidney Aminopeptidase N Expression 
Molecular Pharmaceutics  2014;11(8):2948-2953.
Aminopeptidase N (APN; CD13; EC is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 h postinjection of 99mTc-probestin in a group of 12 UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrate the potential use of 99mTc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.
PMCID: PMC4144757  PMID: 24988047
aminopeptidase N; APN; CD13; alanyl peptidase; probestin; renal pathophysiology; renal cancer; SPECT; imaging
9.  Immuno-PET Imaging of Tumor Endothelial Marker 8 (TEM8) 
Molecular Pharmaceutics  2014;11(11):3996-4006.
Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with 89Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. 89Zr-df–L2mAb was synthesized using a desferioxamine–L2mAb conjugate (df–L2mAb); 125I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. 89Zr-df–L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. 89Zr-df–L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. 125I-L2mAb and 89Zr-df–L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of 89Zr-df–L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. 89Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies 89Zr-df–L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. 89Zr-df–L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, 89Zr-df–L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as 89Zr-df–L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.
PMCID: PMC4224515  PMID: 24984190
immuno-PET imaging; TEM8; anti-TEM8 antibodies; L2; [89Zr]-df−L2mAb; angiogenesis
10.  Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization 
Molecular Pharmaceutics  2014;11(8):2845-2854.
Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids.
PMCID: PMC4144761  PMID: 24978295
acylation; cyclic peptide; cyclization; drug delivery; polyarginine
11.  Design, Synthesis, and Validation of Axl-Targeted Monoclonal Antibody Probe for microPET Imaging in Human Lung Cancer Xenograft 
Molecular Pharmaceutics  2014;11(11):3974-3979.
Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with 64Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe 64Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, 64Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher 64Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, 64Cu-DOTA-h173 uptake in A549 is significantly higher than that of 64Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, 64Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.
PMCID: PMC4224514  PMID: 24978094
Axl receptor; monoclonal antibody; positron emission tomography (PET); lung cancer
12.  Targeting Breast Tumors with pH (Low) Insertion Peptides 
Molecular Pharmaceutics  2014;11(8):2896-2905.
Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors.
PMCID: PMC4123937  PMID: 25004202
imaging; tumor acidity; glucose; lactate dehydrogenase; fluorescence; transgenic mice model
13.  Amphiphilic Nanoparticles Repress Macrophage Atherogenesis: Novel Core/Shell Designs for Scavenger Receptor Targeting and Down-Regulation 
Molecular Pharmaceutics  2014;11(8):2815-2824.
Atherosclerosis, an inflammatory lipid-rich plaque disease is perpetuated by the unregulated scavenger-receptor-mediated uptake of oxidized lipoproteins (oxLDL) in macrophages. Current treatments lack the ability to directly inhibit oxLDL accumulation and foam cell conversion within diseased arteries. In this work, we harness nanotechnology to design and fabricate a new class of nanoparticles (NPs) based on hydrophobic mucic acid cores and amphiphilic shells with the ability to inhibit the uncontrolled uptake of modified lipids in human macrophages. Our results indicate that tailored NP core and shell formulations repress oxLDL internalization via dual complementary mechanisms. Specifically, the most atheroprotective molecules in the NP cores competitively reduced NP-mediated uptake to scavenger receptor A (SRA) and also down-regulated the surface expression of SRA and CD36. Thus, nanoparticles can be designed to switch activated, lipid-scavenging macrophages to antiatherogenic phenotypes, which could be the basis for future antiatherosclerotic therapeutics.
PMCID: PMC4144725  PMID: 24972372
atherosclerosis; amphiphilic macromolecules; scavenger receptor; nanoparticle; macrophages; atherogenesis; oxidized lipoproteins
14.  Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors 
Molecular Pharmaceutics  2014;11(11):3947-3956.
The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-ZEGFR:1907 and 18F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al18F to produce Al18F-NOTA-ZEGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole (18F-CBT) was conjugated to Cys-ZEGFR:1907 to produce 18F-CBT-ZEGFR:1907. Binding affinity and specificity of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al18F-NOTA-ZEGFR:1907 or 18F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al18F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for 18F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al18F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.
PMCID: PMC4218868  PMID: 24972326
affibody; EGFR; PET; 18F; NOTA; CBT
15.  iTEP Nanoparticle-Delivered Salinomycin Displays an Enhanced Toxicity to Cancer Stem Cells in Orthotopic Breast Tumors 
Molecular Pharmaceutics  2014;11(8):2703-2712.
Salinomycin (Sali) has selective toxicity to cancer stem cells (CSCs), a subpopulation of cancer cells that have been recently linked with tumor multidrug resistance (MDR). To utilize its selective toxicity for cancer therapy, we sought to devise a nanoparticle (NP) carrier to deliver Sali to solid tumors through the enhanced permeability and retention effect and, hence, to increase its exposure to CSCs. First, hydrophobic Sali was conjugated to a hydrophilic, immune-tolerant, elastin-like polypeptide (iTEP); the amphiphilic iTEP–Sali conjugates self-assemble into NPs. Next, free Sali was encapsulated into the NPs alone or with two additives, N,N-dimethylhexylamine (DMHA) and α-tocopherol. The coencapsulation significantly improved the loading efficiency and release profile of Sali. The resulting NPs of the coencapsulation, termed as iTEP–Sali NP3s, have an in vitro release half-life of 4.1 h, four times longer than iTEP–Sali NP2s, the NPs that have encapsulated Sali only. Further, the NP3 formulation increases the plasma area under curve and the tumor accumulation of Sali by 10 and 2.4 times, respectively. Lastly, these improved pharmacokinetic and tumor accumulation profiles are consistent with a boost of CSC-elimination effect of Sali in vivo. In NP3-treated 4T1 orthotopic tumors, the mean CSC frequency is 55.62%, a significant reduction from the mean frequencies of untreated tumors, 75.00%, or free Sali-treated tumors, 64.32%. The CSC-elimination effect of the NP3 can further translate to a delay of tumor growth. Given the role of CSCs in driving tumor MDR and recurrence, it could be a promising strategy to add the NP3 to conventional cancer chemotherapies to prevent or reverse the MDR.
PMCID: PMC4216230  PMID: 24960465
cancer stem cells; salinomycin; iTEP nanoparticle; tumor multidrug resistance; coencapsulation additives
16.  Inhibition of Cancer Cell Proliferation and Breast Tumor Targeting of pHLIP-Monomethyl Auristatin E Conjugates 
Molecular pharmaceutics  2015;12(4):1250-1258.
Localized delivery is vital for the successful development of novel and effective therapeutics for the treatment of cancer. The targeting and delivery described herein is based on the pH(Low) Insertion Peptide (pHLIP), a unique delivery peptide that can selectively target tumors in mice and translocate and release cargo molecules intra-cellularly based solely on the low extracellular pH intrinsic to cancer cells. In this study, we investigate the efficacy of pHLIP to target and deliver the highly potent and clinically validated microtubule inhibitor monomethyl auristatin E (MMAE) to cancer cells and breast tumors. We show that pHLIP-MMAE conjugates induce a potent cytotoxic effect (> 90% inhibition of cell growth) in a concentration- and pH-dependent manner after only 2-hour incubation without any apparent disruption of the plasma membrane. pHLIP-MMAE conjugates exhibit between an 11 and 144-fold higher anti-proliferative effect at low pH than at physiological pH, and a pronounced pH-dependent cytotoxicity as compared to free drug. Furthermore, we demonstrate that a pHLIP-MMAE drug conjugate effectively targets triple negative breast tumor xenografts in mice. These results indicate pHLIP-based auristatin conjugates may have an enhanced therapeutic window as compared to free drug, providing a targeting mechanism to attenuate systemic toxicity.
PMCID: PMC4476257  PMID: 25741818
17.  Pluronics and MDR Reversal: An Update 
Molecular Pharmaceutics  2014;11(8):2566-2578.
Multidrug resistance (MDR) remains one of the biggest obstacles for effective cancer therapy. Currently there are only few methods that are available clinically that are used to bypass MDR with very limited success. In this review we describe how MDR can be overcome by a simple yet effective approach of using amphiphilic block copolymers. Triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), arranged in a triblock structure PEO-PPO-PEO, Pluronics or “poloxamers”, raised a considerable interest in the drug delivery field. Previous studies demonstrated that Pluronics sensitize MDR cancer cells resulting in increased cytotoxic activity of Dox, paclitaxel, and other drugs by 2–3 orders of magnitude. Pluronics can also prevent the development of MDR in vitro and in vivo. Additionally, promising results of clinical studies of Dox/Pluronic formulation reinforced the need to ascertain a thorough understanding of Pluronic effects in tumors. These effects are extremely comprehensive and appear on the level of plasma membranes, mitochondria, and regulation of gene expression selectively in MDR cancer cells. Moreover, it has been demonstrated recently that Pluronics can effectively deplete tumorigenic intrinsically drug-resistant cancer stem cells (CSC). Interestingly, sensitization of MDR and inhibition of drug efflux transporters is not specific or selective to Pluronics. Other amphiphilic polymers have shown similar activities in various experimental models. This review summarizes recent advances of understanding the Pluronic effects in sensitization and prevention of MDR.
PMCID: PMC4122590  PMID: 24950236
Pluronic; cancer drug resistance; mitochondria; Pgp; cancer stem cells; lipid rafts; plasma membrane
18.  Insulin Receptor Antibody–Sulfamidase Fusion Protein Penetrates the Primate Blood–Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type A Cells 
Molecular Pharmaceutics  2014;11(8):2928-2934.
Mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase, cause accumulation of lysosomal inclusion bodies in the brain of children born with mucopolysaccharidosis type IIIA, also called Sanfilippo type A syndrome. Enzyme replacement therapy with recombinant SGSH does not treat the brain because the enzyme is a large molecule drug that does not cross the blood–brain barrier (BBB). A BBB-penetrating form of SGSH was produced by re-engineering the enzyme as an IgG fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb domain of the HIRMAb–SGSH fusion protein acts as a molecular Trojan horse to ferry the fused enzyme across the BBB. The HIRMAb–SGSH was produced in stably transfected host cells and purified to homogeneity by protein A chromatography. The fusion protein reacted with antibodies against either human IgG or SGSH on Western blotting. High affinity binding to the HIR was retained following SGSH fusion to the HIRMAb, with an EC50 of 0.33 ± 0.05 nM in an HIR binding ELISA. The SGSH enzyme activity of the HIRMAb–SGSH fusion protein was 4712 ± 388 units/mg protein based on a two-step fluorometric enzyme assay. The HIRMAb–SGSH was taken up by lysosomes in MPSIIIA fibroblasts, and treatment of these cells with the fusion protein caused an 83% reduction in sulfate incorporation into lysosomal glycosoaminoglycans. The HIRMAb–SGSH fusion protein was radiolabeled with the [125I]-Bolton–Hunter reagent and injected intravenously in the Rhesus monkey. The brain uptake of the fusion protein was high, ∼1% injected dose/brain. Calculations, based on this level of brain uptake, suggest normalization of brain SGSH enzyme activity is possible following administration of therapeutic doses of the fusion protein. These studies describe a novel IgG–SGSH fusion protein that is a new noninvasive treatment of the brain in MPS type IIIA.
PMCID: PMC4137762  PMID: 24949884
blood−brain barrier; lysosomal storage; sulfamidase; fusion protein; drug delivery
19.  Clathrin-Mediated Endocytosis Is Impaired in Type A–B Niemann–Pick Disease Model Cells and Can Be Restored by ICAM-1-Mediated Enzyme Replacement 
Molecular Pharmaceutics  2014;11(8):2887-2895.
Drugs often use endocytosis to achieve intracellular delivery, either by passive uptake from the extracellular fluid or by active targeting of cell surface features such as endocytic receptors. An example is enzyme replacement therapy, a clinically practiced treatment for several lysosomal storage diseases where glycosylated recombinant enzymes naturally target the mannose-6-phosphate receptor and are internalized by clathrin mediated endocytosis (CME). However, lysosomal substrate accumulation, a hallmark of these diseases, has been indirectly linked to aberrant endocytic activity. These effects are poorly understood, creating an obstacle to therapeutic efficiency. Here we explored endocytic activity in fibroblasts from patients with type A Niemann–Pick disease, a lysosomal storage disease characterized by acid sphingomyelinase (ASM) deficiency. The uptake of fluid phase markers and clathrin-associated ligands, formation of endocytic structures, and recruitment of intracellular clathrin to ligand binding sites were all altered, demonstrating aberrant CME in these cells. Model polymer nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1), which are internalized by a clathrin-independent route, enhanced the intracellular delivery of recombinant ASM more than 10-fold compared to free enzyme. This strategy reduced substrate accumulation and restored clathrin endocytic activity to wild-type levels. There appears to be a relationship between lysosomal storage and diminished CME, and bypassing this pathway by targeting ICAM-1 may enhance future therapies for lysosomal storage diseases.
PMCID: PMC4144747  PMID: 24949999
type A−B Niemann−Pick disease; clathrin endocytosis; CAM-mediated endocytosis; ICAM-1-targeted nanocarriers; enzyme replacement therapy
20.  In Vivo Tumor Vasculature Targeted PET/NIRF Imaging with TRC105(Fab)-Conjugated, Dual-Labeled Mesoporous Silica Nanoparticles 
Molecular Pharmaceutics  2014;11(11):4007-4014.
Multifunctional mesoporous silica nanoparticles (MSN) with well-integrated multimodality imaging properties have generated increasing research interest in the past decade. However, limited progress has been made in developing MSN-based multimodality imaging agents to image tumors. We describe the successful conjugation of, copper-64 (64Cu, t1/2 = 12.7 h), 800CW (a near-infrared fluorescence [NIRF] dye), and TRC105 (a human/murine chimeric IgG1 monoclonal antibody) to the surface of MSN via well-developed surface engineering procedures, resulting in a dual-labeled MSN for in vivo targeted positron emission tomography (PET) imaging/NIRF imaging of the tumor vasculature. Pharmacokinetics and tumor targeting efficacy/specificity in 4T1 murine breast tumor-bearing mice were thoroughly investigated through various in vitro, in vivo, and ex vivo experiments. Dual-labeled MSN is an attractive candidate for future cancer theranostics.
PMCID: PMC4218929  PMID: 24937108
vasculature targeting; positron emission tomography (PET) imaging; near-infrared fluorescence (NIRF) imaging; mesoporous silica nanoparticle (MSN)
21.  Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic 
Molecular pharmaceutics  2012;9(9):2669-2676.
Our previous studies have demonstrated that a generation 5 dendrimer (G5) conjugated with both folic acid (FA) and methotrexate (MTX) has a higher chemotherapeutic index than MTX alone. Despite this, batch-to-batch inconsistencies in the number of FA and MTX molecules linked to each dendrimer led to conjugate batches with varying biological activity, especially when scaleup synthesis was attempted. Since the MTX is conjugated through an ester linkage, there were concerns that biological inconsistency could also result from serum esterase activity and differential bioavailability of the targeted conjugate. In order to resolve these problems, we undertook a novel approach to synthesize a polyvalent G5–MTXn conjugate through click chemistry, attaching the MTX to the dendrimer through an esterase-stable amide linkage. Surface plasmon resonance binding studies show that a G5–MTX10 conjugate synthesized in this manner binds to the FA receptor (FR) through polyvalent interaction showing 4300-fold higher affinity than free MTX. The conjugate inhibits dihydrofolate reductase, and induces cytotoxicity in FR-expressing KB cells through FR-specific cellular internalization. Thus, the polyvalent MTX on the dendrimer serves the dual role as a targeting molecule as well as a chemotherapeutic drug. The newly synthesized G5–MTXn conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy.
PMCID: PMC4457335  PMID: 22827500
dendrimer; nanoparticle; methotrexate; drug delivery; cancer
22.  Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release 
Molecular pharmaceutics  2014;11(8):2734-2744.
Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems.
PMCID: PMC4457381  PMID: 25020033
endosomal escape; gene therapy; nanoparticle; RNA interference; siRNA
23.  Novel Bifunctional Cyclic Chelator for 89Zr Labeling–Radiolabeling and Targeting Properties of RGD Conjugates 
Molecular Pharmaceutics  2015;12(6):2142-2150.
Within the last years 89Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting αvß3 integrins were synthesized using different conjugation strategies and labeled with 89Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [89Zr]FSC-RGD conjugates or [89Zr]triacetylfusarinine C (TAFC). Quantitative 89Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [89Zr]DFO, [89Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [89Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of 89Zr-based PET imaging agents.
PMCID: PMC4453016  PMID: 25941834
fusarinine C; triacetylfusarinine C; 89Zr; RGD peptide; positron emission tomography (PET)
24.  The design and applications of bispecific heterodimers: molecular imaging and beyond 
Molecular pharmaceutics  2014;11(6):1750-1761.
Ligand-based molecular imaging probes have been designed with high affinity and specificity for monitoring biological process and responses. Single target recognition by traditional probes can limit their applicability for disease detection and therapy as synergistic action between disease mediators and different receptors are often involved in disease progression. Consequently, probes that can recognize multiple targets should demonstrate higher targeting efficacy and specificity than their mono-specific peers. This concept has been validated by multiple bispecific heterodimer-based imaging probes with promising results in several animal models. This review summarizes the design strategies for bispecific peptide and antibody-based heterodimers and their applications in molecular targeting and imaging. The design and application of bispecific heterodimer-conjugated nanomaterials are also discussed.
PMCID: PMC4051252  PMID: 24738564
molecular imaging; heterobivalent ligands; bispecific antibodies; dual targeting; cancer; positron emission tomography (PET)
25.  Targeting to Endothelial Cells Augments the Protective Effect of Novel Dual Bioactive Antioxidant/Anti-Inflammatory Nanoparticles 
Molecular Pharmaceutics  2014;11(7):2262-2270.
Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies.
PMCID: PMC4086738  PMID: 24877560
inflammation; oxidative stress; SOD mimetic; nanocarrier; ROS; tocopherol; cytokines; targeted drug delivery; nanoparticles; antioxidants/anti-inflammatory; tocopherol/vitamin E; endothelial cells

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