Ande Bao

Novel multifunctional theranostic liposome drug delivery system: Construction, characterization, and multimodality MR, near-infrared fluorescent, and nuclear imaging

Liposomes are effective lipid nanoparticle drug delivery systems, which can also be functionalized with noninvasive 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 premanufactured liposomes were composed of DSPC/cholesterol/Gd-DOTA-DSPE/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 postinserted into the premanufactured liposomes. Doxorubicin could be effectively postloaded into the multifunctional liposomes. The multifunctional doxorubicin-liposomes could also be stably radiolabeled with (99m)Tc or (64)Cu 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 noninvasive multimodality NIR fluorescent, MR, SPECT, and PET imaging of their in vivo behavior and capitalizing on the inherent advantages of each modality.

In vivo PET imaging and biodistribution of radiolabeled gold nanoshells in rats with tumor xenografts.

Here we report the radiolabeling of gold nanoshells (NSs) for PET imaging in rat tumor model. A conjugation method was developed to attach NSs with the radionuclide, (64)Cu. The resulting conjugates showed good labeling efficiency and stability in PBS and serum. The pharmacokinetics of (64)Cu-NS and the controls ((64)Cu-DOTA and (64)Cu-DOTA-PEG2K) were determined in nude rats with a head and neck squamous cell carcinoma xenograft by radioactive counting. Using PET/CT imaging, we monitored the in vivo distribution of (64)Cu-NS and the controls in the tumor-bearing rats at various time points after their intravenous injection. PET images of the rats showed accumulation of (64)Cu-NSs in the tumors and other organs with significant difference from the controls. The organ biodistribution of rats at 46h post-injection was analyzed by radioactive counting and compared between the (64)Cu-NS and the controls. Different clearance kinetics was indicated. Neutron activation analysis (NAA) of gold concentration was performed to quantify the amount of NSs in major tissues of the dosed rats and the results showed similar distribution. Overall, PET images with (64)Cu had good resolution and therefore can be further applied to guide photothermal treatment of cancer.

Radioactive liposomes: Radioactive liposomes

Many methods of labeling liposomes with both diagnostic and therapeutic radionuclides have been developed since the initial discovery of liposomes 40 years ago. Diagnostic radiolabels can be used to track nanometer-sized liposomes in the body in a quantitative fashion. This article reviews the basic methods of single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging and labeling of liposomes with single photon and dual photon positron emission radionuclides. Examples of the use of these diagnostic imaging agents will be shown. The ability to track the uptake of liposomes in humans and research animals on a whole body basis is providing researchers with an excellent tool for developing liposome-based drug delivery agents. The attachment of therapeutic radionuclides to liposomes also has great promise in cancer therapy. Recent developments in the use of liposomes carrying therapeutic radionuclides for cancer therapy will also be reviewed. Many of the radiolabeling and tracking technologies developed for nanosized liposomes will also be useful for the imaging and tracking of other nanoparticles.

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.

Effect of intratumoral administration on biodistribution of 64Cu-labeled nanoshells

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Intraoperative 186Re-Liposome Radionuclide Therapy in a Head and Neck Squamous Cell Carcinoma Xenograft Positive Surgical Margin Model

Purpose: Positive surgical margins in advanced head and neck squamous cell carcinoma (HNSCC) have a well-documented association with an increased risk of locoregional recurrence and significantly poorer survival. Traditionally, unresectable tumor is treated with postoperative radiotherapy and/or chemotherapy. However, these therapeutic options can delay treatment and increase toxicity. The potential value of intraoperative injection of liposomal therapeutic radionuclides as a locoregional, targeted therapy in unresectable advanced HNSCC was assessed in a nude rat xenograft positive surgical margin model. Experimental Design: The therapeutic effects of β-emission rhenium-186 (186Re) carried by liposomes into the tumor remnants in a nude rat squamous cell carcinoma xenograft model were studied. Following the partial resection of tumor xenografts, the animals were intratumorally injected with 186Re-labeled or unlabeled (control) neutrally charged or positively charged 100-nm-diameter liposomes. Tumor size, body weight, hematology, and toxicity were monitored for 35 days posttherapy. Results: The neutral (n = 4) and cationic (n = 4) liposome control groups showed an increase in tumor growth of 288.0 ± 37.3% and 292.2 ± 133.7%, respectively, by day 15. The 186Re-neutral-liposome group (n = 8) and the 186Re-cationic-liposome group (n = 8) presented with an average final tumor volume of 25.6 ± 21.8% and 28.5 ± 32.2%, respectively, at the end of the study (day 35). All groups showed consistent increases in body weight. No significant systemic toxicity was observed in any of the animals. Conclusions: With excellent tumor suppression and minimal side-effect profile, the intraoperative use of liposomal therapeutic radionuclides may play a role in the management of positive surgical margins in advanced HNSCC.

Rhenium-186 liposomes as convection-enhanced nanoparticle brachytherapy for treatment of glioblastoma

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 than 80 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 ((186)Re)-liposomes. To better characterize the potential delivery, toxicity, and efficacy of the highly specific activity of (186)Re-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 (186)Re-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 (χ(2)[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 (χ(2)[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 (186)Re-liposomes convection-enhanced delivery in glioma.

Image-guided interventional therapy for cancer with radiotherapeutic nanoparticles

One of the major limitations of current cancer therapy is the inability to deliver tumoricidal agents throughout the entire tumor mass using traditional intravenous administration. Nanoparticles carrying beta-emitting therapeutic radionuclides that are delivered using advanced image-guidance have significant potential to improve solid tumor therapy. The use of image-guidance in combination with nanoparticle carriers can improve the delivery of localized radiation to tumors. Nanoparticles labeled with certain beta-emitting radionuclides are intrinsically theranostic agents that can provide information regarding distribution and regional dosimetry within the tumor and the body. Image-guided thermal therapy results in increased uptake of intravenous nanoparticles within tumors, improving therapy. In addition, nanoparticles are ideal carriers for direct intratumoral infusion of beta-emitting radionuclides by convection enhanced delivery, permitting the delivery of localized therapeutic radiation without the requirement of the radionuclide exiting from the nanoparticle. With this approach, very high doses of radiation can be delivered to solid tumors while sparing normal organs. Recent technological developments in image-guidance, convection enhanced delivery and newly developed nanoparticles carrying beta-emitting radionuclides will be reviewed. Examples will be shown describing how this new approach has promise for the treatment of brain, head and neck, and other types of solid tumors.

Chemoradionuclide Therapy with 186Re-labeled Liposomal Doxorubicin in Combination with Radiofrequency Ablation for Effective Treatment of Head and Neck Cancer in a Nude Rat Tumor Xenograft Model

PURPOSE To determine the therapeutic efficacy of rhenium 186 ((186)Re)-labeled PEGylated liposomal doxorubicin ((186)Re-liposomal doxorubicin) in combination with radiofrequency (RF) ablation of human head and neck squamous cell carcinoma (HNSCC) xenograft in nude rats. MATERIALS AND METHODS This investigation was approved by the animal care committee. Sixty nude rats with subcutaneously implanted HNSCC xenografts (six per group) were treated with (a) RF ablation (70 °C for 5 minutes), (b) PEGylated liposomes, (c) liposomal doxorubicin, (d) (186)Re-PEGylated liposomes (1295 MBq/kg), (e) (186)Re-liposomal doxorubicin (555 MBq/kg), (f) PEGylated liposomes plus RF ablation, (g) liposomal doxorubicin plus RF ablation, (h) (186)Re-PEGylated liposomes plus RF ablation, or (i) (186)Re-liposomal doxorubicin plus RF ablation. Six rats did not receive any treatment (control group). Tumor uptake in (186)Re therapy groups was monitored with small-animal single photon emission computed tomography for 5 days. Therapeutic efficacy was monitored for 6 weeks with measurement of tumor volume, calculation of the percentage injected dose of fluorine 18 fluorodeoxyglucose (FDG) in tumor from small-animal positron emission tomography (PET) images, and determination of viable tumor volume at histopathologic examination. Significant differences between groups were determined with analysis of variance. RESULTS The average tumor volume (± standard deviation) on the day of therapy was 1.32 cm(3) ± 0.17. At 6 weeks after therapy, control of tumor growth was better with (186)Re-liposomal doxorubicin than with liposomal doxorubicin alone (tumor volume, 2.26 cm(3) ± 0.89 vs 5.43 cm(3) ± 0.93, respectively; P < .01). The use of RF ablation with liposomal doxorubicin and (186)Re-liposomal doxorubicin further improved tumor control (tumor volume, 2.05 cm(3) ± 1.36 and 1.49 cm(3) ± 1.47, respectively). The tumor growth trend correlated with change in percentage of injected dose of FDG in tumor for all groups (R(2) = 0.85, P < .001). Viable tumor volume was significantly decreased in the group treated with (186)Re-liposomal doxorubicin plus RF ablation (0.54 cm(3) ± 0.38; P < .001 vs all groups except (186)Re-liposomal doxorubicin alone). CONCLUSION Triple and dual therapies had an observable trend ((186)Re-liposomal doxorubicin plus RF ablation > (186)Re-liposomal doxorubicin > liposomal doxorubicin plus RF ablation > liposomal doxorubicin) of improved tumor growth control and decreased viable tumor compared with other therapies. FDG PET could be used as a noninvasive surrogate marker for tumor growth and viability in this tumor model.

Setup and characterization of a human head and neck squamous cell carcinoma xenograft model in nude rats

OBJECTIVE: To develop and characterize a new head and neck cancer animal model. STUDY DESIGN: A human head and neck squamous cell carcinoma (HNSCC) xenograft model in nude rats was established via subcutaneous inoculation of a human-origin HNSCC cell line, SCC-4. The tumor was evaluated for growth characteristics, pathologic features by hematoxylin-eosin (HE) staining, and immunohistochemistry of epidermal growth factor receptor (EGFR). 2-[18F] fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET) imaging characteristics were studied too. RESULTS: A new HNSCC animal model was successfully established. Tumor sizes reached about 1 cm3 on day 15 after tumor cell inoculation. HE staining pathology has confirmed that this tumor is a typical SCC. EGFR immunohistochemistry demonstrated this tumor model to be strongly EGFR positive. 18F-FDG PET study has shown that 18F-FDG accumulated in tumors. CONCLUSIONS: This study has demonstrated that this tumor model is an appropriate HNSCC tumor model for animal studies on HNSCC.