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1.  Modulation of oxidative stability of haemoglobin inside liposome-encapsulated haemoglobin 
Journal of microencapsulation  2012;30(5):471-478.
The major hurdle in the formulation of liposome-encapsulated haemoglobin (LEH) is the oxidation of haemoglobin (Hb) into methaemoglobin during storage and after administration. In order to reduce this oxidative degradation, we tested various reducing conditions in the presence of catalase. We found that at 37°C more than 50% of Hb oxidized to methaemoglobin within 24 h, whereas in presence of catalase, the oxidation was significantly reduced. The effect of catalase was further enhanced by a reduction mixture containing β-NAD, d-glucose, adenine, inosine, MgCl2, KCl, KH2PO4 and Na2HPO4−, only 14% methaemoglobin was generated in the presence of catalase and reduction mixture (CRM). Contrary to the expectation, glutathione, deferoxamine and homocysteine enhanced Hb oxidation. The presence of CRM inside liposomes (250 nm) significantly decreased Hb oxidation. The results suggest that catalase and a well-defined mixture of co-factors may help control Hb oxidation for improvement in the functional life of LEH.
doi:10.3109/02652048.2012.752535
PMCID: PMC3696053  PMID: 23231644
haemoglobin; transfusion; methaemoglobin; oxygen carriers; liposomes; catalase
2.  A Novel Multifunctional Theranostic Liposome Drug Delivery System: Construction, Characterization, and Multimodality MR, Near-infrared Fluorescent and Nuclear Imaging 
Bioconjugate chemistry  2012;23(6):1322-1332.
Liposomes are effective lipid nanoparticle drug delivery systems, which can also be functionalized with non-invasive multimodality imaging agents with each modality providing distinct information and having synergistic advantages in diagnosis, monitoring of disease treatment, and evaluation of liposomal drug pharmacokinetics. We designed and constructed a multifunctional theranostic liposomal drug delivery system, which integrated multimodality magnetic resonance (MR), near-infrared (NIR) fluorescent and nuclear imaging of liposomal drug delivery, and therapy monitoring and prediction. The pre-manufactured liposomes were composed of DSPC/cholesterol/Gd-DOTADSPE/DOTA-DSPE with the molar ratio of 39:35:25:1 and having ammonium sulfate/pH gradient. A lipidized NIR fluorescent tracer, IRDye-DSPE, was effectively post-inserted into the pre-manufactured liposomes. Doxorubicin could be effectively post-loaded into the multifunctional liposomes. The multifunctional doxorubicin-liposomes could also be stably radiolabeled with 99mTc or 64Cu for single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging, respectively. MR images displayed the high resolution micro-intratumoral distribution of the liposomes in squamous cell carcinoma of head and neck (SCCHN) tumor xenografts in nude rats after intratumoral injection. NIR fluorescent, SPECT and PET images also clearly showed either the high intratumoral retention or distribution of the multifunctional liposomes. This multifunctional drug carrying liposome system is promising for disease theranostics allowing non-invasive multimodality NIR fluorescent, MR, SPECT and PET imaging of their in vivo behavior and capitalizing on the inherent advantages of each modality.
doi:10.1021/bc300175d
PMCID: PMC3435449  PMID: 22577859
Multifunctional liposomes; Theranostics; Drug delivery; Multimodality imaging; Intratumoral retention and distribution; Nanoparticles
3.  Feasibility of Eradication of Breast Cancer Cells Remaining in Post-Lumpectomy Cavity and Draining Lymph Nodes Following Intracavitary Injection of Radioactive Immunoliposomes 
Molecular pharmaceutics  2012;9(9):2513-2522.
Most diagnosed early stage breast cancer cases are treated by lumpectomy and adjuvant radiation therapy, which significantly decreases the locoregional recurrence but causes inevitable toxicity to normal tissue. By using a technique of preparing liposomes carrying technetium-99m (99mTc), rhenium-186 (186Re), or rhenium-188 (188Re) radionuclides, as well as chemotherapeutic agents, or their combination, for cancer therapy with real time image-monitoring of pharmacokinetics and prediction of therapy effect, this study investigated the potential of a novel targeted focal radiotherapy with low systemic toxicity using radioactive immunoliposomes to treat both the surgical cavity and draining lymph nodes in a rat breast cancer xenograft positive surgical margin model. Immunoliposomes modified with either panitumumab (anti-EGFR), or bevacizumab (anti-VEGF) were remote loaded with 99mTc diagnostic radionuclide, and injected into the surgical cavity of female nude rats with positive margins post lumpectomy. Locoregional retention and systemic distribution of 99mTc-immunoliposomes were investigated by nuclear imaging, stereofluorescent microscopic imaging and gamma counting. Histopathological examination of excised draining lymph nodes was performed. The locoregional retention of 99mTc-immunoliposomes in each animal was influenced by the physiological characteristics of surgical site of individual animals. Panitumumab- and bevacizumab-liposome groups had higher intracavitary retention compared with the control liposome groups. Draining lymph node uptake was influenced by both the intracavitary radioactivity retention level and metastasis status. Panitumumab-liposome group had higher accumulation on the residual tumor surface and in the metastatic lymph nodes. Radioactive liposomes that were cleared from the cavity were metabolized quickly and accumulated at low levels in vital organs. Therapeutic radionuclide-carrying specifically targeted panitumumab- and bevacizumab- liposomes have increased potential compared to non-antibody targeted liposomes for post-lumpectomy focal therapy to eradicate remaining breast cancer cells inside the cavity and draining lymph nodes with low systemic toxicity.
doi:10.1021/mp300132f
PMCID: PMC3444252  PMID: 22894603
breast cancer; targeted therapy; immunoliposomes; intracavitary injection; metastasis
4.  Rhenium-186 liposomes as convection-enhanced nanoparticle brachytherapy for treatment of glioblastoma 
Neuro-Oncology  2012;14(4):416-425.
Although external beam radiation is an essential component to the current standard treatment of primary brain tumors, its application is limited by toxicity at doses more than80 Gy. Recent studies have suggested that brachytherapy with liposomally encapsulated radionuclides may be of benefit, and we have reported methods to markedly increase the specific activity of rhenium-186 (186Re)–liposomes. To better characterize the potential delivery, toxicity, and efficacy of the highly specific activity of 186Re-liposomes, we evaluated their intracranial application by convection-enhanced delivery in an orthotopic U87 glioma rat model. After establishing an optimal volume of 25 µL, we observed focal activity confined to the site of injection over a 96-hour period. Doses of up to 1850 Gy were administered without overt clinical or microscopic evidence of toxicity. Animals treated with 186Re-liposomes had a median survival of 126 days (95% confidence interval [CI], 78.4–173 days), compared with 49 days (95% CI, 44–53 days) for controls. Log-rank analysis between these 2 groups was highly significant (P = .0013) and was even higher when 100 Gy was used as a cutoff (P < .0001). Noninvasive luciferase imaging as a surrogate for tumor volume showed a statistically significant separation in bioluminescence by 11 days after 100 Gy or less treatment between the experimental group and the control animals (χ2[1, N= 19] = 4.8; P = .029). MRI also supported this difference in tumor size. Duplication of tumor volume differences and survival benefit was possible in a more invasive U251 orthotopic model, with clear separation in bioluminescence at 6 days after treatment (χ2[1, N= 9] = 4.7; P = .029); median survival in treated animals was not reached at 120 days because lack of mortality, and log-rank analysis of survival was highly significant (P = .0057). Analysis of tumors by histology revealed minimal areas of necrosis and gliosis. These results support the potential efficacy of the highly specific activity of brachytherapy by 186Re-liposomes convection-enhanced delivery in glioma.
doi:10.1093/neuonc/nos060
PMCID: PMC3309864  PMID: 22427110
brachytherapy; glioblastoma; glioma; liposomes; nanoparticles
5.  Novel Asparagine-Derived Lipid Enhances Distearoylphosphatidylcholine Bilayer Resistance to Acidic Conditions 
A novel asparagine-derived lipid analogue (ALA11,17) bearing a tetrahydropyrimidinone head group and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol% or greater ALA11,17 in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA11,17 in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA11,17-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA11,17/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA11,17 is a promising additive for modulating phosphatidylcholine lipid bilayer properties.
doi:10.1021/la105085k
PMCID: PMC3539164  PMID: 21413760
Tetrahydropyrimidinone; lipid; DSPC liposomes; nanostructures; multilamellar vesicles; scanning electron microscopy
6.  Chemoradionuclide Therapy with 186Re-Labeled Liposomal Doxorubicin: Toxicity, Dosimetry, and Therapeutic Response 
Abstract
This study was performed to determine the maximum tolerated dose (MTD) and therapeutic effects of rhenium-186 (186Re)-labeled liposomal doxorubicin (Doxil), investigate associated toxicities, and calculate radiation absorbed dose in head and neck tumor xenografts and normal organs. Doxil and control polyethylene glycol (PEG)-liposomes were labeled using 186Re-N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (BMEDA) method. Tumor-bearing rats received either no therapy (n=6), intravenous Doxil (n=4), or escalating radioactivity of 186Re-Doxil (185–925 MBq/kg) or 186Re-PEG-liposomes (1110–1665 MBq/kg) and were monitored for 28 days. Based on body weight loss and systemic toxicity, MTD for 186Re-Doxil and 186Re-PEG-liposomes were established at injected radioactivity/body weight of 740 and 1480 MBq/kg, respectively. 186Re-injected radioactivity/body weight for therapy studies was determined to be 555 MBq/kg for 186Re-Doxil and 1295 MBq/kg for 186Re-PEG-liposomes. All groups recovered from their body weight loss, leucopenia, and thrombocytopenia by 28 days postinjection. Normalized radiation absorbed dose to tumor was significantly higher for 186Re-Doxil (0.299±0.109 Gy/MBq) compared with 186Re-PEG-liposomes (0.096±0.120 Gy/MBq) (p<0.05). In a separate therapy study, tumor volumes were significantly smaller for 186Re-Doxil (555 MBq/kg) compared with 186Re-PEG-liposomes (1295 MBq/kg) (p<0.01) at 42 days postinjection. In conclusion, combination chemoradionuclide therapy with 186Re-Doxil has promising potential, because good tumor control was achieved with limited associated toxicity.
doi:10.1089/cbr.2010.0948
PMCID: PMC3189008  PMID: 21834653
186Re; liposomal doxorubicin; MTD; head and neck cancer; radionuclide therapy; imaging
7.  Effect of intratumoral administration on biodistribution of 64Cu-labeled nanoshells 
Video abstract
Video
Background
Gold nanoshells are excellent agents for photothermal ablation cancer therapy and are currently under clinical trial for solid tumors. Previous studies showed that passive delivery of gold nanoshells through intravenous administration resulted in limited tumor accumulation, which represents a major challenge for this therapy. In this report, the impact of direct intratumoral administration on the pharmacokinetics and biodistribution of the nanoshells was systematically investigated.
Methods
The gold nanoshells were labeled with the radionuclide, copper-64 (64Cu). Intratumoral infusion of 64Cu-nanoshells and two controls, ie, 64Cu-DOTA (1,4,7,10-tetraazaciclododecane- 1,4,7,10-tetraacetic acid) and 64Cu-DOTA-PEG (polyethylene glycol), as well as intravenous injection of 64Cu-nanoshells were performed in nude rats, each with a head and neck squamous cell carcinoma xenograft. The pharmacokinetics was determined by radioactive counting of serial blood samples collected from the rats at different time points post-injection. Using positron emission tomography/computed tomography imaging, the in vivo distribution of 64Cu-nanoshells and the controls was monitored at various time points after injection. Organ biodistribution in the rats at 46 hours was analyzed by radioactive counting and compared between the different groups.
Results
The resulting pharmacokinetic curves indicated a similar trend between the intratumorally injected agents, but a significant difference with the intravenously injected 64Cu-nanoshells. Positron emission tomography images and organ biodistribution results on rats after intratumoral administration showed higher retention of 64Cu-nanoshells in tumors and less concentration in other healthy organs, with a significant difference from the controls. It was also found that, compared with intravenous injection, tumor concentrations of 64Cu-nanoshells improved substantially and were stable at 44 hours post-injection.
Conclusion
There was a higher intratumoral retention of 64Cu-nanoshells and a lower concentration in other healthy tissues, suggesting that intratumoral administration is a potentially better approach for nanoshell-based photothermal therapy.
doi:10.2147/IJN.S30699
PMCID: PMC3356223  PMID: 22619558
gold nanoshells; intratumoral administration; positron emission tomography; biodistribution
8.  Post-Lumpectomy Focal Brachytherapy for Simultaneous Treatment of Surgical Cavity and Draining Lymph Nodes 
Purpose
The primary objective was to investigate a novel focal brachytherapy technique using lipid nanoparticle (liposome)-carried β-emitting radionuclides (rhenium-186 [186Re]/rhenium-188 [188Re]) to simultaneously treat the post-lumpectomy surgical cavity and draining lymph nodes.
Methods and Materials
Cumulative activity distributions in lumpectomy cavity and lymph nodes were extrapolated from small animal imaging and human lymphoscintigraphy data. Absorbed dose calculations were performed for lumpectomy cavities with spherical and ellipsoidal shapes and lymph nodes within human subjects using the dose point kernel convolution method.
Results
Dose calculations showed that therapeutic dose levels within the lumpectomy cavity wall can cover 2 and 5 mm depths for 186Re- and 188Re-liposomes, respectively. The absorbed doses at 1 cm sharply decreased to only 1.3 – 3.7% of the doses at 2 mm for 186Re-liposomes and 5 mm for 188Re-liposomes. Concurrently, the draining sentinel lymph nodes would receive a high focal therapeutic absorbed dose, while the average dose to 1 cm of surrounding tissue received < 1% of that within the nodes.
Conclusions
Focal brachytherapy using 186Re/188Re-liposomes was theoretically shown capable of simultaneously treating the lumpectomy cavity wall and draining sentinel lymph nodes with high absorbed doses while significantly lowering dose to surrounding healthy tissue. In turn, this allows for dose escalation to regions of higher probability of containing residual tumor cells following lumpectomy while reducing normal tissue complications.
doi:10.1016/j.ijrobp.2010.05.062
PMCID: PMC3031997  PMID: 20864271
Breast cancer; Lymph node; Focal brachytherapy; Liposome; Radionuclide
9.  Bone marrow-targeted liposomal carriers 
Expert opinion on drug delivery  2011;8(3):317-328.
Introduction
Bone marrow targeted drug delivery systems appear to offer a promising strategy for advancing diagnostic, protective, and/or therapeutic medicine for the hematopoietic system. Liposome technology can provide a drug delivery system with high bone marrow targeting that is mediated by specific phagocytosis in bone marrow.
Area covered
This review focuses on a bone marrow specific liposome formulation labeled with technetium-99m (99mTc). Interspecies differences in bone marrow distribution of the bone marrow targeted formulation are emphasized. This review provides a liposome technology to target bone marrow. In addition, the selection of proper species for the investigation of bone marrow targeting is suggested.
Expert opinion
It can be speculated that the bone marrow macrophages have a role in the delivery of lipids to the bone marrow as a source of energy and for membrane biosynthesis or in the delivery of fat soluble vitamins for hematopoiesis. This homeostatic system offers a potent pathway to deliver drugs selectively into bone marrow tissues from blood. High selectivity of the present BMT-liposome formulation for bone marrow suggests the presence of an active and specific mechanism, but specific factors affecting the uptake of the bone marrow MPS are still unknown. Further investigation of this mechanism will increase our understanding of factors required for effective transport of agents to the bone marrow, and may provide an efficient system for bone marrow delivery for therapeutic purposes.
doi:10.1517/17425247.2011.553218
PMCID: PMC3076608  PMID: 21275831
liposomes; bone marrow targeting; drug delivery; biodistribution; scintigraphy; mononuclear phagocyte system
10.  Post-lumpectomy intracavitary retention and lymph node targeting of 99mTc-encapsulated liposomes in nude rats with breast cancer xenograft 
Liposomes are recognized drug delivery systems with tumor-targeting capability. In addition, therapeutic or diagnostic radionuclides can be efficiently loaded into liposomes. This study investigated the feasibility of utilizing radiotherapeutic liposomes as a new post-lumpectomy radiotherapy for early-stage breast cancer by determining the locoregional retention and systemic distribution of liposomes radiolabeled with technetium-99m (99mTc) in an orthotopic MDA-MB-231 breast cancer xenograft nude rat model. To test this new brachytherapy approach, a positive surgical margin lumpectomy model was set up by surgically removing the xenograft and deliberately leaving a small tumor remnant in the surgical cavity. Neutral, anionic, and cationic surface-charged fluorescent liposomes of 100 and 400 nm diameter were manufactured and labeled with 99mTc-BMEDA. Locoregional retention and systemic distribution of 99mTc-liposomes injected into the post-lumpectomy cavity were determined using non-invasive nuclear imaging, ex vivo tissue gamma counting and fluorescent stereomicroscopic imaging. The results indicated that 99mTc-liposomes were effectively retained in the surgical cavity (average retention was 55.7 ± 24.2% of injected dose for all rats at 44 h post-injection) and also accumulated in the tumor remnant (66.9 ± 100.4%/g for all rats). The majority of cleared 99mTc was metabolized quickly and excreted into feces and urine, exerting low radiation burden on vital organs. In certain animals 99mTc-liposomes significantly accumulated in the peripheral lymph nodes, especially 100 nm liposomes with anionic surface charge. The results suggest that post-lumpectomy intracavitary administration of therapeutic radionuclides delivered by 100-nm anionic liposome carrier is a potential therapy for the simultaneous treatment of the surgical cavity and the draining lymph nodes of early-stage breast cancer.
doi:10.1007/s10549-010-1309-x
PMCID: PMC3184211  PMID: 21181436
Breast cancer; Radioactive liposomes; Intracavitary; Brachytherapy; Locoregional retention; Lymph node
11.  Preclinical multimodality phantom design for quality assurance of tumor size measurement 
BMC Medical Physics  2011;11:1.
Background
Evaluation of changes in tumor size from images acquired by ultrasound (US), computed tomography (CT) or magnetic resonance imaging (MRI) is a common measure of cancer chemotherapy efficacy. Tumor size measurement based on either the World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST) is the only imaging biomarker for anti-cancer drug testing presently approved by the United States Food and Drug Administration (FDA). The aim of this paper was to design and test a quality assurance phantom with the capability of monitoring tumor size changes with multiple preclinical imaging scanners (US, CT and MRI) in order to facilitate preclinical anti-cancer drug testing.
Methods
Three phantoms (Gammex/UTHSCSA Mark 1, Gammex/UTHSCSA Mark 2 and UTHSCSA multimodality tumor measurement phantom) containing tumor-simulating test objects were designed and constructed. All three phantoms were scanned in US, CT and MRI devices. The size of test objects in the phantoms was measured from the US, CT and MRI images. RECIST, WHO and volume analyses were performed.
Results
The smaller phantom size, simplified design and better test object CT contrast of the UTHSCSA multimodality tumor measurement phantom allowed scanning of the phantom in preclinical US, CT and MRI scanners compared with only limited preclinical scanning capability of Mark 1 and Mark 2 phantoms. For all imaging modalities, RECIST and WHO errors were reduced for UTHSCSA multimodality tumor measurement phantom (≤1.69 ± 0.33%) compared with both Mark 1 (≤ -7.56 ± 6.52%) and Mark 2 (≤ 5.66 ± 1.41%) phantoms. For the UTHSCSA multimodality tumor measurement phantom, measured tumor volumes were highly correlated with NIST traceable design volumes for US (R2 = 1.000, p < 0.0001), CT (R2 = 0.9999, p < 0.0001) and MRI (R2 = 0.9998, p < 0.0001).
Conclusions
The UTHSCSA multimodality tumor measurement phantom described in this study can potentially be a useful quality assurance tool for verifying radiologic assessment of tumor size change during preclinical anti-cancer therapy testing with multiple imaging modalities.
doi:10.1186/1756-6649-11-1
PMCID: PMC3206432  PMID: 21958653
12.  Radiobiological characterization of post-lumpectomy focal brachytherapy with lipid nanoparticle-carried radionuclides 
Physics in medicine and biology  2011;56(3):703-719.
Post-operative radiotherapy has commonly been used for early stage breast cancer to treat residual disease. The primary objective of this work was to characterize, through dosimetric and radiobiological modeling, a novel focal brachytherapy technique which uses direct intracavitary infusion of β-emitting radionuclides (186Re/188Re) carried by lipid nanoparticles (liposomes). Absorbed dose calculations were performed for a spherical lumpectomy cavity with a uniformly injected activity distribution using a dose point kernel convolution technique. Radiobiological indices were used to relate predicted therapy outcome and normal tissue complication of this technique with equivalent external beam radiotherapy treatment regimens. Modeled stromal damage was used as a measure of the inhibition of the stimulatory effect on tumor growth driven by the wound healing response. A sample treatment plan delivering 50 Gy at a therapeutic range of 2.0 mm for 186Re-liposomes and 5.0 mm for 188Re-liposomes takes advantage of the dose delivery characteristics of the β-emissions, providing significant EUD (58.2 Gy and 72.5 Gy for 186Re and 188Re, respectively) with a minimal NTCP (0.046%) of the healthy ipsilateral breast. Modeling of kidney BED and ipsilateral breast NTCP showed that large injected activity concentrations of both radionuclides could be safely administered without significant complications.
PMCID: PMC3169207  PMID: 21299006
13.  Interventional Therapy of Head and Neck Cancer with Lipid Nanoparticle-Carried Rhenium-186 Radionuclide 
Purpose
Minimally invasive interventional cancer therapy of drug-carrying lipid nanoparticles (liposomes) via convection enhanced delivery generally applied by the use of an infusion pump can increase intratumoral drug concentration and retention while facilitating broad distribution throughout solid tumors. We investigated the utility of liposome-carrying β-emitting radionuclides to treat head and neck cancer in nude rats by direct intratumoral infusion.
Methods
Four groups of nude rats were subcutaneously inoculated with human tongue cancer cells. After tumors reached an average size of 1.6 cm3, the treatment group received an intratumoral infusion of liposomal rhenium-186 (186Re) (185 MBq (5 mCi)/cm3 tumor). Three control groups were intratumorally infused with either, 1) unlabeled liposomes, 2) unencapsulated 186Re-perrhenate, or 3) unencapsulated intermediate 186Re-compound (186Re-BMEDA). In vivo distribution of 186Re-activity was measured by planar gamma camera imaging. Tumor therapy and toxicity were assessed by measurements of tumor size, body weight, and hematology.
Results
Average tumor volume of the 186Re-liposome group on post-treatment day-14 decreased to 87.7±20.1%, while tumor volumes increased to 395.0% - 514.4% on average in other three groups (P<0.001 vs 186Re-liposome group). 186Re-liposomes provided much higher intratumoral retention of 186Re-activity, resulting in an average tumor radiation absorbed dose of 526.3±93.3 Gy, whereas 186Re-perrhenate and 186Re-BMEDA groups had only 3.3±1.2 and 13.4±9.2 Gy tumor doses respectively. No systemic toxicity was observed.
Conclusion
Liposomal 186Re effectively treated the head and neck cancer with minimal side effects after convection enhanced interventional delivery. These results suggest the potential of liposomal 186Re for clinical application in interventional therapy of cancer.
doi:10.1016/j.jvir.2010.02.027
PMCID: PMC2910224  PMID: 20478719
14.  Isolation and Characterization of a Metastatic Hybrid Cell Line Generated by ER Negative and ER Positive Breast Cancer Cells in Mouse Bone Marrow 
PLoS ONE  2011;6(6):e20473.
Background
The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior.
Methods
ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.
Results
The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.
Conclusions
Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.
doi:10.1371/journal.pone.0020473
PMCID: PMC3106006  PMID: 21673810
15.  Integrin αvβ3-targeted gold nanoshells augment tumor vasculature-specific imaging and therapy 
Purpose
Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvβ3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvβ3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy.
Methods
Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS–RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS–RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs.
Results
ELISA and cell binding assay confirmed the binding affinity of NS–RGDfK to integrin αvβ3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis.
Conclusion
The results presented in this paper set the stage for the advancement of integrin αvβ3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.
doi:10.2147/IJN.S15479
PMCID: PMC3058535  PMID: 21423588
nanoparticle; cyclo(RGDfK); cancer; thermal ablation
16.  186Re-Liposomal Doxorubicin (Doxil): In Vitro Stability, Pharmacokinetics, Imaging and Biodistribution in a Head and Neck Squamous Cell Carcinoma Xenograft Model 
Nuclear medicine and biology  2009;36(5):515-524.
doi:10.1016/j.nucmedbio.2009.02.004
PMCID: PMC2696057  PMID: 19520292
Rhenium-186; Doxil; biodistribution; pharmacokinetics; SPECT/CT; liposome; nanoparticle
17.  Caspase-2 Deficiency Enhances Aging-Related Traits in Mice 
Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.
doi:10.1016/j.mad.2006.11.030
PMCID: PMC1828128  PMID: 17188333
caspase-2; maximum lifespan; bone; hair growth; fat

Results 1-17 (17)