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Figure S1. Drug dose-dependent enhancement of γ-H2AX and Rad51 expression using NanoOlaparib; Figure S2. Representative confocal microscopy images of Rad51 and Polθ immunostaining 30 minutes following irradiation.

Cerebrovascular abnormality is linked to Alzheimer’s disease and related dementias (ADRDs). ApoE-Ɛ4 (APOE4) is known to play a critical role in neurovascular dysfunction, however current medical imaging technologies are limited in quantification. This cross-sectional study tested the feasibility of a recently established imaging modality, quantitative ultra-short time-to-echo contrast-enhanced magnetic resonance imaging (QUTE-CE MRI), to identify small vessel abnormality early in development of human APOE4 knock-in female rat (TGRA8960) animal model. At 8 months, 48.3% of the brain volume was found to have significant signal increase (75/173 anatomically segmented regions; q

The use of PARP inhibitors in combination with radiotherapy is a promising strategy to locally enhance DNA damage in tumors. Here we show that radiation-resistant cells and tumors derived from a Pten/Trp53-deficient mouse model of advanced prostate cancer are rendered radiation sensitive following treatment with NanoOlaparib, a lipid-based injectable nanoformulation of olaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to NanoOlaparib alone. In animals, twice-weekly intravenous administration of NanoOlaparib results in significant tumor growth inhibition, whereas previous studies of oral olaparib as monotherapy have shown no therapeutic efficacy. When NanoOlaparib is administered prior to radiation, a single dose of radiation is sufficient to triple the median mouse survival time compared to radiation only controls. Half of mice treated with NanoOlaparib + radiation achieved a complete response over the 13-week study duration. Using ferumoxytol as a surrogate nanoparticle, MRI studies revealed that NanoOlaparib enhances the intratumoral accumulation of systemically administered nanoparticles. NanoOlaparib-treated tumors showed up to 19-fold higher nanoparticle accumulation compared to untreated and radiation-only controls, suggesting that the in vivo efficacy of NanoOlaparib may be potentiated by its ability to enhance its own accumulation. Together, these data suggest that NanoOlaparib may be a promising new strategy for enhancing the radiosensitivity of radiation-resistant tumors lacking BRCA mutations, such as those with PTEN and TP53 deletions. Mol Cancer Ther; 16(7); 1279–89. ©2017 AACR.

The rising prevalence and severity of antibiotic-resistant biofilm infections poses an alarming threat to public health worldwide. Here, biocompatible multi-compartment nanocarriers were synthesized to contain both hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) and the hydrophilic antibiotic methicillin for the treatment of medical device-associated infections. SPION co-encapsulation was found to confer unique properties, enhancing both nanocarrier relaxivity and magneticity compared to individual SPIONs. These iron oxide-encapsulating polymersomes (IOPs) penetrated 20 μm thick Staphylococcus epidermidis biofilms with high efficiency following the application of an external magnetic field. Three-dimensional laser scanning confocal microscopy revealed differential bacteria death as a function of drug and SPION loading. Complete eradication of all bacteria throughout the biofilm thickness was achieved using an optimized IOP formulation containing 40 μg/mL SPION and 20 μg/mL of methicillin. Importantly, this formulation was selectively toxic towards methicillin-resistant biofilm cells but not towards mammalian cells. These novel iron oxide-encapsulating polymersomes demonstrate that it is possible to overcome antibiotic-resistant biofilms by controlling the positioning of nanocarriers containing two or more therapeutics.

Encapsulating bacteria within constrained microenvironments can promote the manifestation of specialized behaviors. Using double-emulsion droplet-generating microfluidic synthesis, live Bacillus subtilis bacteria were encapsulated in a semi-permeable membrane composed of poly(ethylene glycol)-b-poly(D,L-lactic acid) (mPEG-PDLLA). This polymer membrane was sufficiently permeable to permit exponential bacterial growth, metabolite-induced gene expression, and rapid biofilm growth. The biodegradable microparticles retained structural integrity for several days and could be successfully degraded with time or sustained bacterial activity. Microencapsulated B. subtilis successfully captured and contained sodium selenite added outside the polymersomes, converting the selenite into elemental selenium nanoparticles that were selectively retained inside the polymer membrane. This remediation of selenium using polymersomes has high potential for reducing the toxicity of environmental selenium contamination, as well as allowing selenium to be harvested from areas not amenable to conventional waste or water treatment.

Poly(ADP-ribose) polymerase (PARP) inhibitors have been majorly utilized in cancers with BRCA1/2 mutations that have deficiencies in the homologous recombination (HR) DNA repair pathway. Traditionally, PARP inhibitors have been administered through oral route in the clinic but they have resulted in poor bioavailability, low tumor accumulation and high systemic toxicity. Moreover, tumors develop resistance to PARP inhibitors necessitating the development of strategies to re-sensitize these resistant tumors. To overcome these limitations, nanoparticle drug formulation has demonstrated a great potential to increase the drug concentration and accumulation at tumor sites with low systemic toxicities. Here we report the development of several nanoformulations of PARP inhibitors and observe their efficacies by systemic administration to treat various cancer diseases. We used two FDA approved PARP inhibitors (Olaparib or Talazoparib) for nanoformulations of NanoOlaparib and NanoTalazoparib, decorated with EpCAM or EGFR antibody, and tested on breast, ovarian, prostate and lung cancer with or without irradiation. In HR deficient models, the nanoformulations showed significant inhibition of tumor cell growth in vitro and in vivo. Above all, combination of PARP inhibitor with irradiation resulted in greater efficacy in lung and prostate cancer models. To overcome PARP inhibitor resistance, we formulated nanoparticles, NanoDinaciclib of cyclin-dependent kinase 12 (CDK12) inhibitor (dinaciclib) and used in combination with nanoformulation of PARP inhibitor to arrest the tumor growth in animal model. Our current research reveals that the use of CDK12 inhibitor in PARP resistant tumors further arrests tumor growth compared with monotherapy alone. Further studies will help clarify to achieve clear understanding of the mechanism of action CDK12 inhibitor to determine the best way to use PARP inhibitors beyond HR deficiency. Citation Format: Bijay Singh, Shicheng Yang, Paige Baldwin, Anne van de Ven, Srinivas Sridhar. Nanoformulations of PARP and CDK inhibitors for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A104. doi:10.1158/1535-7163.TARG-19-A104

The data in this article provide detail regarding the rat brain atlas measurements discussed in our research article, “Quantitative vascular neuroimaging of the rat brain using superparamagnetic nanoparticles: New insights on vascular organization and brain function” (Gharagouzloo et al., 2017) [1]. This article provides datasets of quantitative cerebral blood volume (qCBV) measurements across 173 regions of the rat brain in 11 healthy rats. State-changes from this baseline during isoflurane and CO2 administration are provided for all regions and all animals.

A method called Quantitative Ultra-Short Time-to-Echo Contrast Enhanced (QUTE-CE) Magnetic Resonance Imaging (MRI) which utilizes superparamagnetic iron oxide nanoparticles (SPIONs) as a contrast agent to yield positive contrast angiograms with high clarity and definition is applied to the whole live rat brain. QUTE-CE MRI intensity data are particularly well suited for measuring quantitative cerebral blood volume (qCBV). A global map of qCBV in the awake resting-state with unprecedented detail was created via application of a 3D MRI rat brain atlas with 173 segmented and annotated brain areas. From this map we identified two distributed, integrated neural circuits showing the highest capillary densities in the brain. One is the neural circuitry involved with the primary senses of smell, hearing and vision and the other is the neural circuitry of memory. Under isoflurane anesthesia, these same circuits showed significant decreases in qCBV suggesting a role in consciousness. Neural circuits in the brainstem associated with the reticular activating system and the maintenance of respiration, body temperature and cardiovascular function showed an increase in qCBV with anesthesia. During awake CO2 challenge, 84 regions showed significant increases relative to an awake baseline state. This CO2 response provides a measure of cerebral vascular reactivity and regional perfusion reserve with the highest response measured in the somatosensory cortex. These results demonstrate the utility of QUTE-CE MRI for qCBV analysis and offer a new perspective on brain function and vascular organization.

Metallic nanoparticles with a high atomic number release Auger electrons in response to external beam X-ray radiation. When these nanoparticles are selectively delivered to tumors, they have the potential to locally enhance the effects of radiation therapy. Optimizing the therapeutic efficacy of these nanoparticles, however, remains a challenging and time-consuming task. Here we describe three different assays that can be used to experimentally quantify and optimize the in vitro therapeutic efficacy of nanoparticle-mediated X-ray radiation enhancement. These include an IC50 extended dose response curve, clonogenic cell survival assay, and immunoblotting. Collectively, these assays provide information about whether a given nanoparticle provides radiosensitization, the extent of the radiosensitization, and the potential mechanism of radiosensitization.

The use of PARP inhibitors in combination with radiation therapy is a promising strategy to locally enhance DNA-damage in tumors. Here we show that radiation-resistant cells and tumors derived from a Pten/Trp53-deficient mouse model of advanced prostate cancer are rendered radiation-sensitive following treatment with NanoOlaparib, a lipid-based injectable nanoformulation of Olaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to NanoOlaparib alone. In animals, twice-weekly intravenous administration of NanoOlaparib results in significant tumor growth inhibition, whereas previous studies of oral Olaparib as monotherapy have shown no therapeutic efficacy. When NanoOlaparib is administered prior to radiation, a single dose of radiation is sufficient to triple the median mouse survival time compared to radiation only controls. Half of mice treated with NanoOlaparib + radiation achieved a complete response over the 13 week study duration. Using ferumoxytol as a surrogate nanoparticle, MRI studies revealed that NanoOlaparib enhances the intratumoral accumulation of systemically administered nanoparticles. NanoOlaparib-treated tumors showed up to 19-fold higher nanoparticle accumulation compared to untreated and radiation-only controls, suggesting that the in vivo efficacy of NanoOlaparib may be potentiated by its ability to enhance its own accumulation. Together, this data suggests that NanoOlaparib may be a promising new strategy for enhancing the radiosensitivity of radiation-resistant tumors lacking BRCA mutations, such as those with PTEN and TP53 deletions.

Nanoparticles for cancer therapy and imaging are designed to accumulate in the diseased tissue by exploiting the Enhanced Permeability and Retention (EPR) effect. This limits their size to about 100 nm. Here, using intravital microscopy and elemental analysis, we compare the in vivo localization of particles with different geometries and demonstrate that plateloid particles preferentially accumulate within the tumor vasculature at unprecedented levels, independent of the EPR effect. In melanoma-bearing mice, 1000 × 400 nm plateloid particles adhered to the tumor vasculature at about 5% and 10% of the injected dose per gram organ (ID/g) for untargeted and RGD-targeted particles respectively, and exhibited the highest tumor-to-liver accumulation ratios (0.22 and 0.35). Smaller and larger plateloid particles, as well as cylindroid particles, were more extensively sequestered by the liver, spleen, and lungs. Plateloid particles appeared well-suited for taking advantage of hydrodynamic forces and interfacial interactions required for efficient tumoritropic accumulation, even without using specific targeting ligands.

Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of 'losing sight of the forest for the trees'. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of "-omic" technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon "-omic" technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology "snapshot" of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to "self-correct" in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success.

The rapidly diminishing number of effective antibiotics that can be used to treat infectious diseases and associated complications in a physician's arsenal is having a drastic impact on human health today. This study explored the development and optimization of a polymersome nanocarrier formed from a biodegradable diblock copolymer to overcome bacterial antibiotic resistance. Here, polymersomes were synthesized containing silver nanoparticles embedded in the hydrophobic compartment, and ampicillin in the hydrophilic compartment. Results showed for the first time that these silver nanoparticle-embedded polymersomes (AgPs) inhibited the growth of Escherichia coli transformed with a gene for ampicillin resistance (bla) in a dose-dependent fashion. Free ampicillin, AgPs without ampicillin, and ampicillin polymersomes without silver nanoparticles had no effect on bacterial growth. The relationship between the silver nanoparticles and ampicillin was determined to be synergistic and produced complete growth inhibition at a silver-to-ampicillin ratio of 1 : 0.64. In this manner, this study introduces a novel nanomaterial that can effectively treat problematic, antibiotic-resistant infections in an improved capacity which should be further examined for a wide range of medical applications.

Introduction: The objective of this project is to study the biological pathways activated in irradiated pancreatic cancer cells pre-treated with gold nanoparticles. Metallic nanoparticles emit Auger electrons and photoelectrons upon exposure to X-rays. When selectively delivered to tumors, these nanoparticles can locally enhance the effects of radiation therapy. Previous in vitro work has primarily studied the effectiveness of nanoparticle-enhanced therapy, without elucidating the underlying biological mechanisms. Understanding the biological mechanisms (such as changes in gene expression) of how nanoparticles enhance radiation therapy can help in the further design of more effective nanoparticles. Methods: Gold nanoparticle sensitization was tested using a human based pancreatic cancer cell line, Capan-1. Nanoparticle toxicity was tested using a combination of MTS and Alamar Blue assays after 24 hours of treatment. Gene expression was tested using immunofluorescence and western blot techniques. Cells were pretreated with 0.8 mg gold nanoparticles 24 hours prior to radiation. Cells were then harvested at various time points to determine how gene expression changes over time. Radiosensitization was carried out using a clonogic cell survival assay where cells were pretreated with 0.8 mg gold nanoparticles 24 hours prior to radiation and then re-plated at various densities 4 hours post radiation, and allowed to incubate for 14 days. Statistical differences were determined using ANOVA followed by student t-tests. Results: The MTS and Alamar blue assays showed low gold nanoparticle toxicity towards cells up to a dose of 1mg for Capan-1 cells. Capan-1 cells pretreated with 0.8 mg gold nanoparticles for 24 hours prior to radiation showed lower cell survival than cells only treated with radiation. Western blots and immunofluorescence data showed that certain genes, such as γ-H2AX, were upregulated when pretreated with gold nanoparticles and irradiated compared to cells treated with radiation alone. It was also shown that γ-H2AX expression peaked around 30 minutes post radiation and then returned to basal levels after 24 hours. Conclusions: Capan-1 cells experience more DNA damage when pretreated with gold nanoparticles than when only treated with radiation. γ-H2AX, which is a DNA damage-related protein, was upregulated in the combined dose showing that there was more DNA damage in the combined treatment. It was also shown that protein and gene expression analysis needs to be carried out at various time points post radiation to obtain a better understanding of the mechanisms involved in radiation induced damage to pancreatic cancer cells. Supported by NSF-DGE- 0965843 and CA188833-02. Citation Format: Autumn D. Paro, Ilan Shanmugam, Anne van de Ven, Rajiv Kumar, Thomas J. Webster, Srinivas Sridhar. Biological mechanisms involved in nanoparticle-enhanced radiation therapy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B44.

Sustained localized delivery of cancer therapeutics is a safe and effective unique option for local control of tumors. Here we report a novel biodegradable implant with the capability to encapsulate different therapeutics, molecular agents, or nanoparticles for local intratumoral delivery. We have successfully demonstrated in vivo the delivery of PARP inhibitor Talazoparib to treat Brca1-mutated cancers and Docetaxel to treat localized or recurring prostate cancers. This one-time intratumoral injection provides a safe vehicle for the sustained release of PARP inhibitor Talazoparib and chemotherapeutic Docetaxel in contrast to low bioavailability and toxicity associated with oral or systemic delivery. Methods: Biodegradable implants of 1-2mm length and 0.8mm diameter were loaded with ~50μg Talazoparib for BRCA1-mutated breast cancer studies and ~500μg Docetaxel (DTX) for prostate cancer studies. The implants were characterized in vitro using SEM and HPLC, and the release kinetic studies were carried out in PBS buffer (pH 6.0) at 37°C. The IC50's were determined using an MTS assay in breast cancer cell lines derived from Brca1 Co/Co; MMTV-Cre; p53+/−mice, W0069 and W780, and human-derived prostate cancer, PC3. In vivo studies were carried out in Brca1 Co/Co; MMTV-Cre; p53+/− spontaneous tumored mice for breast cancer studies. Subcutaneous PC3 tumors were xenografted in nude mice. Prostate cancer studies were done with and without radiation. Drug-loaded implants were injected once intratumorally using an 18G brachytherapy needle. Results: The release profile of the drug from the implant in buffer showed a highly sustained release for multiple weeks at therapeutically relevant doses for both docetaxel and Talazoparib loading implants. Breast cancer cell lines W0069 and W780 were highly sensitive to Talazoparib, most likely due to Brca1 mutation. Following a one-time intratumoral implantation of Talazoparib, tumors reduced in size by an average of 50%, while untreated tumors increased ~5X in size. Talazoparib dosing appeared to be well tolerated by the mice. Docetaxel implants proved to be an effective method for prostate cancer in vivo with no significant weight loss observed. The local docetaxel spacer group showed sustained tumor inhibition compared to empty implants and an equivalent DTX dose given systemically. At 40 days 89% survival was observed for mice treated with localized DTX implants compared with 0% in all other treatment groups. Histology samples were taken from sacrificed mice and immunohistochemistry are currently underway. Conclusions: Sustained local release of therapeutically relevant doses of Talazoparib and Docetaxel were observed in vitro and in vivo. Therapeutics-loaded implants represent a novel delivery route that are well-tolerated. Sustained release of Talazoparib appears to amplify the therapeutic efficacy of PARP inhibition in BRCA1 mutated breast cancers and sustained release of Docetaxel is an effective chemotherapy option alone or in combination with radiation therapy. These results laid a strong foundation for the use of localized biodegradable implants for the treatment of breast and prostate cancer. This work was supported by the Army- W81XWH-14-1-0092, Breast Cancer Research Foundation and Northeastern University–Dana Farber Cancer Institute collaborative grant. Citation Format: Jodi Belz, Noelle Castilla Ojo, Paige Baldwin, Rajiv Kumar, Anne van de Ven, Karen Liby, Robert Cormack, Mike Makrigiorgos, Srinivas Sridhar4. Sustained release of PARP inhibitor Talazoparib and chemotherapeutic Docetaxel from modified brachytherapy spacers for treatment of breast and prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B30.

Introduction: We have developed a new method of Quantitative MRI named QUTE-CE MRI that yields images of the vasculature with unparalleled clarity and definition and is quantitative. QUTE-CE MRI can produce contrast enhanced magnetic resonance angiograms (CE-MRA) using super paramagnetic iron-oxide nanoparticle (SPION), including the FDA approved ferumoxytol, with high contrast in cardiovascular, cerebral, and tumor imaging. Based upon principles of magnetic nanoparticle interactions with neighboring water molecules, the method achieves robust, reproducible results by utilizing rapid signal acquisition at ultra-short time-to-echo (UTE) to produce positive-contrast images with pure T1 weighting and little T2* decay. The spoiled gradient echo equation (SPGR) is used to transform UTE intensities directly into concentration using experimentally determined relaxivity constants and image acquisition parameters. Methods: All animal experiments were conducted in accordance with the Northeastern University Division of Laboratory Animal Medicine and Institutional Animal Care and Use Committee. MRI images were obtained at ambient temperature (∼25°C) using a Bruker Biospec 7.0T/20-cm USR horizontal magnet (Bruker, Billerica, Massachusetts, USA) equipped with a 20-G/cm magnetic field gradient insert (ID =12 cm, Bruker) and the same quadrature 300 MHz, 30 mm Mouse MRI coil was used for all in vivo work as previously utilized for mouse experiments in Section 3.8 (Animal Imaging Research, LLC, Holden, Massachusetts, USA). PC 3 cells were injected into the right flank of immunocompromised FoxNu1 mice (n=5, Charles River Laboratories). After tumors reached about 0.5-1.0cm3, animals underwent three separate imaging sessions: Session 1 - pre-contrast T1, T2 and QUTE-CE measurements, Session 2 - immediate post-contrast QUTE-CE measurement and Session 3 - 24h post-contrast T1, T2 and QUTE-CE measurements. For contrast, 100μl of ferumoxytol diluted to 6mg/ml was injected i.v. to render a blood concentration of ~200μg/ml Fe (2x clinical dose). Results: Contrary to more standard MRI techniques, QUTE-CE pre-contrast images render a nearly homogenous signal with a Gaussian distribution in the tumor. The immediate post-contrast images render the vasculature clearly and skew the distribution of voxels within the whole tumor to the left, however also increases the overall mean of the signal intensity because the movement of voxels within the tumor is to the right, leaving a long bright tail with the brightest voxels represented by those containing 100% blood. 24h after the initial administration of ferumoxytol the vasculature is no longer visible, but the locations within the tumor that have passively accumulated SPIONs resulting from the EPR effect becomes apparent. While the distribution of voxels within the tumor becomes less skewed, the overall shape is still slightly skewed to the left and the mean of the distribution has moved to the right. Nanoparticle accumulation in the post-contrast image is heterogeneous and unambiguous. Angiography and TBV in tumors Assuming a partial 2-volume model of blood and tissue, we determine the tumor blood volume (TBV) across the entire tumor volume. The resultant TBV heatmaps show a clear range of TBV values are apparent, delineating areas of the tissue with high contrast in regard to overall vascular health, including apparently necrotic tissue. Nanoparticle accumulation Next, a unique feature of the methodology to produce high-contrast images of purely T1-weighted signal is employed to unambiguously delineate nanoparticle accumulation in a PC3 subcutaneous tumor model with ferumoxytol accumulation 24 hours after just one dose. From this, contrast efficiency was produced compared to standard techniques with the additional benefit that pre-contrast images are not necessitated. A major advantage of delineating SPION accumulation using QUTE-CE, compared to ΔT2 or ΔT1 imaging, is that the post-contrast image contains sufficient information for nanoparticle localization, eliminating the need for pre-contrast images. Conclusion: QUTE-CE MRI exploits physical principles of magnetic relaxation modulated by SPIONs to achieve quantitative MRI yielding exceptional vascular images. This ability to longitudinally quantify blood pool CA concentration is unique to the QUTE-CE method, and makes QUTE-CE MRI competitive with nuclear imaging. Quantitative tumor blood volume distributions are obtained at short times, while nanoparticle accumulation maps are obtained at long times. QUTE-CE MRI is a new method that can be used to study tumor properties longitudinally. The technique is immediately translatable to the clinic using the FDA approved contrast agent ferumoxytol and is expected to have a major impact on clinical tumor imaging. Work supported by NSF-DGE- 0965843. Citation Format: Gharagouzloo Codi, Ju Qiao, Liam Timms, Anne van de Ven, Srinivas Sridhar. Quantitative tumor imaging using magnetic nanoparticles. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B22.

Prostate cancers with PTEN deletions are promising candidates for DNA repair inhibitors such as olaparib and talazoparib. Here we show that radiation-resistant cells and tumors derived from Ptenpc-/-;Trp53pc-/- mice are rendered radiation-sensitive following pre-treatment with liposomal nanoOlaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to nanoformulated Olaparib alone. In animals, twice-weekly intravenous administration of nanoOlaparib alone results in significant tumor growth inhibition. When nanoOlaparib is administered prior to radiation, we find that a single dose of radiation is sufficient to increase mouse survival time by as much as 10 weeks (study duration = 13 weeks). Using ferumoxytol as a surrogate nanoparticle, magnetic resonance imaging (MRI) studies revealed that nanoOlaparib administration enhances the ability of nanoparticles to accumulate in tumors. Compared to untreated and radiation-only controls, nanoOlaparib-treated tumors showed 18-fold higher nanoparticle accumulation, suggesting that the in vivo efficacy of nanoOlaparib may be potentiated by its ability to enhance its own accumulation in tumors. Citation Format: Anne L. van de Ven, Shifalika Tangutoori, Paige Baldwin, Ju Qiao, Codi Gharagouzloo, Nina Seitzer, John Clohessy, Houari Korideck, G. Mike Makrigiorgos, Robert Cormack, Pier Paolo Pandolfi, Srinivas Sridhar. Prostate cancer pre-treatment with nanoformulated Olaparib overcomes radiation resistance. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B48.

The investigation of microcirculation is a critical task in biomedical and physiological research. In order to monitor human’s condition and develop effective therapies of some diseases, the microcirculation information, such as flow velocity and vessel density, must be evaluated in a noninvasive manner. As one of the tasks of microcirculation investigation, automatic blood cell tracking presents an effective approach to estimate blood flow velocity. Currently, the most common method for blood cell tracking is based on spatiotemporal image analysis, which has lots of limitations, such as the diameter of microvesssels cannot be too larger than blood cells or tracers, cells or tracers should have fixed velocity, and it requires the image with high qualification. In this paper, we propose an optical flow method for automatic cell tracking. The key algorithm of the method is to align an image to its neighbors in a large image collection consisting of a variety of scenes. Considering the method cannot solve the problems in all cases of cell movement, another optical flow method, SIFT (Scale Invariant Feature Transform) flow, is also presented. The experimental results show that both methods can track the cells accurately. Optical flow is specially robust to the case where the velocity of cell is unstable, while SIFT flow works well when there are large displacement of cell between two adjacent frames. Our proposed methods outperform other methods when doing in vivo cell tracking, which can be used to estimate the blood flow directly and help to evaluate other parameters in microcirculation.

Enhanced permeation and retention (EPR) effect, the mechanism by which nanotherapeutics accumulate in tumors, varies in patients based on differences in the tumor and organ microenvironment. Surrogate biomarkers for the EPR effect will aid in selecting patients who will accumulate higher amounts of nanotherapeutics and show better therapeutic efficacy. Our data suggest that the differences in the vascular permeability and pegylated liposomal doxorubicin (PLD) accumulation are tumor type as well as organ-specific and significantly correlated with the relative ratio of MMP-9 to TIMP-1 in the circulation, supporting development of these molecules as biomarkers for the personalization of nanoparticle-based therapy.

Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response.

Sustained localized delivery of cancer therapeutics is a safe and effective unique option for non-metastatic cancers. Here we report a novel biodegradable implant with the capability to encapsulate therapeutics, molecular agents, or nanoparticles for local intratumoral delivery. We have successfully demonstrated in vivo the delivery of PARP inhibitor Talazoparib to treat Brca1-mutated cancers and Docetaxel to treat localized or recurring prostate cancers. This one-time intratumoral injection provides a safe vehicle for the sustained release of PARP inhibitor Talazoparib and chemotherapeutic Docetaxel in contrast to low bioavailability and toxicity associated with oral or systemic delivery. Methods: Biodegradable implants of 1-2mm length and 0.8mm diameter were loaded with ∼50μg Talazoparib (BMN) for BRCA1-mutated breast cancer (BCa) studies and ∼500μg Docetaxel (DTX) for prostate cancer (PCa) studies. Implants were characterized using SEM and HPLC, and release studies were carried out in pH 6.0 PBS buffer at 37°C. The IC50's were determined using an MTS assay in cell lines W0069 and W780 (BCa) and PC3 (PCa). In vivo studies were carried out in Brca1 Co/Co;MMTV-Cre; p53+/− spontaneous tumored mice for BCa studies. Subcutaneous PC3 tumors were xenografted in nude mice. PCa studies were done with and without radiation. Implants were injected once intratumorally using an 18G brachytherapy needle. Results: The release profile of the drug from the implant in buffer showed a highly sustained release for multiple weeks at therapeutically relevant doses for both DTX and BMN loaded implants. BCa cell lines W0069 and W780 were highly sensitive to BMN, most likely due to Brca1 mutation. Following a one-time intratumoral implantation of BMN, tumors reduced in size by an average of 50%, while untreated tumors increased ∼5X in size. BMN dosing appeared to be well tolerated by the mice. DTX implants proved to be an effective method for PCa treatment in vivo with no weight loss observed. The local DTX group showed sustained tumor inhibition compared to empty implants and an equivalent DTX dose given systemically. At 40 days 89% survival was observed for mice treated with localized DTX implants compared with 0% in all other treatment groups. Histology samples were taken from sacrificed mice and immunohistochemistry is currently underway. Conclusions: Sustained local release of therapeutically relevant doses of BMN and DTX were observed in vitro and in vivo. Therapeutics loaded in implants represent a novel delivery modality that is well-tolerated. Sustained release of BMN appears to amplify the therapeutic efficacy of PARP inhibition in BRCA1 mutated breast cancers and sustained release of DTX is an effective chemotherapy option alone or in combination with radiation therapy. These results lay a strong foundation for the use of localized biodegradable implants for the treatment of breast and prostate cancer. Citation Format: Jodi Belz, Noelle Castilla Ojo, Paige Baldwin, Rajiv Kumar, Anne van de Ven, Karen Liby, Robert Cormack, Mike Makrigiorgos, Srinivas Sridhar. Sustained release of PARP inhibitor Talazoparib and chemotherapeutics from biodegradable implants for treatment of breast and prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3900.

Introduction: Poly(ADP-ribose) Polymerase (PARP) plays an important role in a number of DNA repair pathways. PARP inhibitors (PARPi) such as Olaparib and Talazoparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. These drugs are currently only available in oral form which results in limited bioavailability, poor tumor accumulation, and systemic toxicity. Here we report the development of novel nanoformulations of Olaparib and Talazoparib to allow intravenous or intraperitoneal delivery, providing greater bioavailability and tumor accumulation, while limiting systemic toxicities. Methods: Nanoparticle formulations of Olaparib and Talazoparib were synthesized and tested in vitro and in vivo. Short-and long-term dose response with a panel of ovarian cancer cell lines were conducted. These cell lines include KURAMOCHI, SKOV3, OVSAHO, JHOS2, PA1, COV318, 403 and 404, derived from BRCA2-/-, PTEN-/-, TP53mut mice, and 4306 and 4412, developed from conditional LSL-K-rasG12D/+, PTENloxP/loxP mice. Radiosensitization with NanoOlaparib was tested in the radiation resistant prostate cancer cell line FK01, derived from Ptenpc-/-;Trp53pc-/- mice. In vivo, NanoOlaparib was tested in an IP spread model using 404 cells. Animals were treated IP with NanoOlaparib alone, and in combination with cisplatin. Radiosensitization with NanoOlaparib in vivo was tested in a xenograft model using FK01 cells to mimic castration resistant prostate cancer. Animals were treated biweekly with NanoOlaparib before and after radiation treatment. Results: The murine cell lines 403 and 404 were highly sensitive to this treatment due to the mutations in BRCA2, PTEN, and TP53. 4412 and 4306 showed comparable sensitivity, suggesting that a PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. PA1 demonstrated high sensitivity to NanoOlaparib which may be attributed to genetic instability. NanoTalazoparib is more potent than NanoOlaparib, resulting in a similar relationship in cell line sensitivity with overall lower IC50’s. Strong synergistic radiosensitization was observed in FK01 cells with NanoOlaparib. Bioluminescence imaging illustrated that NanoOlaparib administered IP daily resulted in a greater inhibition of tumor growth than those treated with oral Olaparib daily. The FK01 xenografts are highly radioresistant with little difference between untreated and radiation only animals. NanoOlaparib delays tumor growth, while the combination of radiation and NanoOlaparib clearly shrinks tumors. Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully developed for in vitro and in vivo studies. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and prostate cancers. Citation Format: Paige Baldwin, Anders Ohman, Jeremy Thong, Shifalika Tangutoori, Anne van de Ven, Rajiv Kumar, Daniela Dinulescu, Srinivas Sridhar. Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4335.

This manuscript constitutes a review of several innovative biomedical technologies fabricated using the precision and accuracy of silicon micro- and nanofabrication. The technologies to be reviewed are subcutaneous nanochannel drug delivery implants for the continuous tunable zero-order release of therapeutics, multi-stage logic embedded vectors for the targeted systemic distribution of both therapeutic and imaging contrast agents, silicon and porous silicon nanowires for investigating cellular interactions and processes as well as for molecular and drug delivery applications, porous silicon (pSi) as inclusions into biocomposites for tissue engineering, especially as it applies to bone repair and regrowth, and porous silica chips for proteomic profiling. In the case of the biocomposites, the specifically designed pSi inclusions not only add to the structural robustness, but can also promote tissue and bone regrowth, fight infection, and reduce pain by releasing stimulating factors and other therapeutic agents stored within their porous network. The common material thread throughout all of these constructs, silicon and its associated dielectrics (silicon dioxide, silicon nitride, etc.), can be precisely and accurately machined using the same scalable micro- and nanofabrication protocols that are ubiquitous within the semiconductor industry. These techniques lend themselves to the high throughput production of exquisitely defined and monodispersed nanoscale features that should eliminate architectural randomness as a source of experimental variation thereby potentially leading to more rapid clinical translation.

Cancer research has achieved dramatic advances in recent years with modern techniques and aroused extensive efforts to discover effective therapeutic drugs. As complement to time and resource intensive traditional drug development methods, application of computational models for drug pharmacokinetics and pharmacodynamics research has become increasingly popular because of its high efficiency and flexibility. Our work provides a well-designed integrated mechanistic model for developing cancer drugs by incorporating new techniques like PET, CT, and intravital microscopy. This model studies drug distribution and therapeutic effects from a systematic viewpoint at three different yet highly related levels. Firstly, a macro-scale model was established to study time course drug distributions in different organs especially in blood which circulates in whole body and delivers drug to targeted tumor. At the intratumoral level, a coupled 3D tumor growth and angiogenesis model was proposed to elaborately simulate neovasculature formation and calculate regional variations in drug distribution inside solid tumor. The third part focuses on how regional variations in drug distribution affect tumor cell death/proliferation rate by linking therapeutic response to intracellular signaling pathway blockade or alteration, which provides us a high-resolution overview of drug action in the targeted tumor cell. Hence, the first two modules which target the organ level and intratumoral microenvironment are directly coupled to pharmacokinetics analysis which is linked to the last component of the model that analyzes the pharmacodynamic effects at the cellular level. Experimental data for calibrating computational model, like drug distribution, tumor vasculature, drug penetration rate etc., were measured using PET, CT, and high resolution intravital microscopy. We perform simulations to investigate the delivery efficiency and drug efficacy of free drug and nanotherapeutics. Our model successfully predicted optimal drug property and therapy strategy. Finally, model effectiveness was validated by in vivo experimental data.

The breast cancer-associated gene 1 (Brca1) is the most frequently mutated tumor suppressor gene found in familial breast cancers. Mutations of the gene modulate many cellular functions including DNA damage and repair, homologous recombination, cell-cycle regulation, and apoptosis. Poly-ADP-Ribose Polymerase (PARP) inhibitor therapy can produce cell death in cancers with genetic predispositions for impaired DNA repair or transcription pathways such as Brca1 mutants. Here we report a novel biodegradable implant for the local delivery of PARP inhibitor Talazoparib to treat Brca1-mutated cancers. This one-time intratumoral injection provides a safe vehicle for the sustained release of PARP inhibitor Talazoparib in contrast to low bioavailability and toxicity associated with oral delivery. Methods: Biodegradable implants of 1-2mm length and 0.8mm diameter were loaded with ∼50μg Talazoparib. The implants were characterized in vitro using SEM and HPLC, and the release kinetic studies were carried out in PBS buffer (pH 6.0) at 37°C. The IC50 was determined using an MTS assay in breast cancer cell lines derived from Brca1 Co/Co; MMTV-Cre; p53+/−mice. In vivo studies were carried out in Brca1 Co/Co; MMTV-Cre; p53+/− mice. Drug-loaded implants were injected once intratumorally using an 18G brachytherapy needle. Results: In vitro studies The release profile of the drug from the implant in buffer showed a highly sustained release for multiple weeks at therapeutically relevant doses. Breast cancer cell lines W0069 and W780, derived from Brca1 Co/Co; MMTV-Cre; p53+/− mice were highly sensitive to Talazoparib, most likely due to Brca1 mutation. PARP expression was examined via western blot analysis. In vivo studies In vivo studies using sustained drug release implants loaded with Talazoparib were also carried out in Brca1Co/Co;MMTV-Cre;p53+/- genetically engineered mice with 1 or more spontaneous breast tumors. Following a one-time implantation, tumors reduced in size by an average of 50%, while untreated tumors increased ∼5X in size. Talazoparib dosing appeared to be well tolerated by the mice. Histology samples were taken from sacrificed mice and immunohistochemistry are currently underway. Conclusions: Sustained local release of therapeutically relevant doses of Talazoparib was observed in vitro and in vivo. The Talazoparib-loaded implants represent a novel delivery route that was well-tolerated. Sustained release of Talazoparib appears to amplify the therapeutic efficacy of PARP inhibition and is a promising new route for the treatment of highly aggressive breast cancer models. We would like to acknowledge the Breast Cancer Research Foundation. This work was supported by the Army- W81XWH-14-1-0092 and Northeastern University – Dana Farber Cancer Institute collaborative grant. Citation Format: Jodi Belz, Karen Liby, Paige Baldwin, Rajiv Kumar, Anne L. van de Ven, Robert Cormack, Mike Makrigiorgos, Srinivas Sridhar. Sustained release of PARP inhibitor Talazoparib from bioedgradable implant for treatment of BRCA1-mutated breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B42.

Solid tumors develop abnormally at spatial and temporal scales, giving rise to biophysical barriers that impact anti-tumor chemotherapy. This may increase the expenditure and time for conventional drug pharmacokinetic and pharmacodynamic studies. In order to facilitate drug discovery, we propose a mathematical model that couples three-dimensional tumor growth and angiogenesis to simulate tumor progression for chemotherapy evaluation. This application-oriented model incorporates complex dynamical processes including cell- and vascular-mediated interstitial pressure, mass transport, angiogenesis, cell proliferation, and vessel maturation to model tumor progression through multiple stages including tumor initiation, avascular growth, and transition from avascular to vascular growth. Compared to pure mechanistic models, the proposed empirical methods are not only easy to conduct but can provide realistic predictions and calculations. A series of computational simulations were conducted to demonstrate the advantages of the proposed comprehensive model. The computational simulation results suggest that solid tumor geometry is related to the interstitial pressure, such that tumors with high interstitial pressure are more likely to develop dendritic structures than those with low interstitial pressure.

Heterogeneities in the perfusion of solid tumors prevent optimal delivery of nanotherapeutics. Clinical imaging protocols for obtaining patient-specific data have proven difficult to implement. It is challenging to determine which perfusion features hold greater prognostic value and to relate measurements to vessel structure and function. With the advent of systemically administered nanotherapeutics whose delivery is dependent on overcoming diffusive and convective barriers to transport, such knowledge is increasingly important. We describe a framework for the automated evaluation of vascular perfusion curves measured at the single vessel level. Primary tumor fragments, collected from triple-negative breast cancer patients and grown as xenografts in mice, were injected with fluorescence contrast and monitored using intravital microscopy. The time to arterial peak and venous delay, two features whose probability distributions were measured directly from time-series curves, were analyzed using a fuzzy c-mean supervised classifier in order to rank individual tumors according to their perfusion characteristics. The resulting rankings correlated inversely with experimental nanoparticle accumulation measurements, enabling the modeling of nanotherapeutics delivery without requiring any underlying assumptions about tissue structure or function, or heterogeneities contained therein. With additional calibration, these methodologies may enable the investigation of nanotherapeutics delivery strategies in a variety of tumor models.

Chemotherapeutic delivery is generally poor in tumors characterized by rapid perfusion and low blood volume fraction. Nanoparticles can be engineered to overcome abnormal flow conditions to act as intravascular drug depots for the localized delivery of high concentrations of chemotherapeutics. We hypothesized that the ability of multi-stage nanotherapeutics to accumulate in tumors is highly dependent on tumor perfusion and that matching particle parameters to tumor-specific flow parameters can improve the delivery of nanotherapeutics. To test this hypothesis, we measured first-pass perfusion of an intra-arterial bolus of FITC-dextran using intravital microscopy (IVM) in 4 patient-derived breast tumor xenograft (PDX) lines: BCM-2665, BCM-4195, BCM-2147, and BCM-3887. These PDX lines were established directly from patient samples and maintain the triple negative biomarker status of the original patient tumors. Selected for their differing vascular morphologies, these 4 tumor lines yielded distinct, reproducible transport features unique to each tissue. BCM-2665 tumors (n=6) were characterized by regions of dense vascularization interspersed with regions of little to no blood flow. BCM-2147 tumors (n=6) were dominated by large tortuous vessels like those observed in BCM-2665 tumors, but also had a well-developed capillary network interconnecting the large vessels. BCM-3887 tumors (n=6) were characterized by an extensive network of poorly defined, dilated, leaky microvessels. BCM-4195 tumors were uniformly covered with a well-defined, widely spaced microvessel network. The arteriovenous transit time (AVTT) was measured by tracking a bolus of 40kDa FITC-dextran through the tumor vasculature in real-time. Both 2665 and 2147 tumors were rapidly perfused (∼10 - 25 seconds), whereas a majority of 3887 and 4195 tumors required ∼45 seconds or more. The blood volume fraction (BVF) varied by nearly an order of magnitude, with 2665 and 2147 tumors exhibiting the lowest BVF (∼0.11 - 0.12). The tracer elimination constant (Kel), which was measured as a surrogate for tumor permeability using time-lapse IVM images, was an order of magnitude lower for 3887 and 4195 tumors (∼0.11/hr vs. ∼1.17/hr) indicating that these tumors are much more permeable than 2665 and 2147 tumors. Interestingly, flow parameters that adversely impact drug accumulation appear to favor particle accumulation. In clinical studies, tumors characterized by rapid perfusion and low blood volume fraction generally respond poorly to chemotherapy and radiation therapy. We have observed this phenomenon in our cancer models: BCM-2665 and BCM-2147 tumors fail to show growth inhibition at clinically relevant doses of docetaxel, whereas BCM-4195 and BCM-3887 tumors show complete growth inhibition. As such, we believe these tumors will provide an excellent model for studying the relative importance of tumor-specific transport features for improving drug delivery. Citation Format: Anne L. Van de Ven-Moloney, Melissa D. Landis, Lacey A. Burey, Mauro Ferrari, Jenny C. Chang. Assessing the role of tumor vascularity in nanotherapeutics delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3909. doi:10.1158/1538-7445.AM2013-3909

Pegylated liposomal doxorubicin (PLD) for cancer therapy is advantageous over conventional chemotherapy with doxorubicin, because of the preferential delivery of drugs to tumors owing to the enhanced permeation and retention (EPR) effect. The biological barriers include abnormal structure of tumor vessels in heterogeneous tumor microenvironments shall influence the EPR effect and result in heterogeneous tumor perfusion of PLD and therapeutic efficacy. Although 4T1, murine breast cancer cells and 3LL, murine lung cancer cells had similar sensitivity to PLD in vitro, only 4T1 tumors responded to therapy with PLD in vivo. There were no significant differences of the microvessel density and blood perfusion in these tumors. In contrast, PLD extravasated and accumulated into 4T1 tumors significantly more than 3LL tumors, indicating vascular permeability was higher in 4T1 tumors. Coverage of endothelial cells by collagen type IV, which constitutes basement membrane of the vessels, was significantly lower in 4T1 tumors as compared to those in 3LL tumors. Differential analysis of protein expression by 4T1 and 3LL cells in vitro revealed that MMP-9 (collagenase) production was significantly higher in 4T1 cells as compared to 3LL cells. MMP-9 expression was also higher in 4T1 tumors as well as in sera of mice bearing 4T1 tumors as compared to 3LL tumors and sera of mice bearing 3LL tumors and normal mice respectively. Batimastat, MMPs inhibitor, injected in vivo increased the coverage of endothelial cells by basement membrane and abrogated the accumulation of PLD into the 4T1 tumors, indicating MMP-9 can play a pivotal role in controlling the vascular permeability. Interestingly, 4T1 tumors were accumulated with PLD only when tumors were growing in the brain and mammary fat pad, but not in the liver. Coverage of the endothelial cells by basement membrane was significantly higher in the tumors growing in the liver as compared to the other two locations. The levels of TIMP-1, endogenous inhibitor of MMPs, were significantly higher in the mice bearing 4T1 in the liver as compared to the mice bearing the tumors in the other locations. These data indicate the levels as well as balance between MMP-9 and TIMP-1 can determine the vascular permeability to PLD. Therefore the these enzymes in the circulation can serve as surrogate markers for the vascular permeability to PLD and there is a possibility to personalize the therapy by selecting patients who will likely accumulated with PLD into tumors to increase clinical outcome. Citation Format: Kenji Yokoi, Tomonori Tanei, Biana Godin, Anne van de Ven, Jenolyn Alexander, Mauro Ferrari. Tumor type and organ type dependent differences of vascular permeability to pegylated liposomal doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4973. doi:10.1158/1538-7445.AM2013-4973

Liver metastasis occur in 30-70% of patients dying of various malignancies. In the case of metastatic breast cancer, 50% of the patients develop liver metastasis with median survival rate of only a few months and very rare 5-year survival. We hypothesized that the inefficiency in treatment of liver metastasis of breast tumors can be related to biophysical barriers in the tumor microenvironment preventing delivery of therapeutics to tumor loci. Our research focused on physical factors involved in the progression of liver metastasis and design of systems for targeted delivery of therapeutics. Multi-stage vectors (MSV) were previously reported to overcome sequential biophysical barriers and to efficiently carry therapeutic payload to the tumor microenvironment. The first stage porous silicon particles target tumor-associated endothelium or macrophages releasing second stage therapeutic nanoparticles in the close proximity to the tumor. The objectives of this work were to investigate: (1) in vivo barriers associated with drug delivery in breast tumor liver metastasis; (2) in vitro/in vivo interaction of MSV with macrophages; and (3) to perform initial therapeutic efficiency study with MSV loaded with Abraxane (albumin bound paclitaxel). Liver metastases were produced by intra-splenic injected of 4T1 breast cancer cells to balb/C mice. A small incision in the abdomen of the animals was made to expose the liver and the kinetics of distribution of tracers/MSV was recorded. Intravital microscopy was used to observe the distribution of tracers (MW 4 and 30KDa dextrans) and MSV particles. Macrophages and RBC were pre-labeled 4-48 hours prior to the experiment. For therapy experiment, 10-14 days following tumor inoculation mice were randomly divided in three groups: untreated, MSV-Abraxane treated and Abraxane treated. The mice were sacrificed on day 7 following the treatment and the number of metastasis and liver weight were recorded. From the intravital microscopy studies, it appears that liver metastasis of 4T1 tumors have insufficient functional vascularization, which can be the reason for inability to efficiently treat metastatic loci. Dextran −4KDs with nominal diameter of 2-3nm (similar to unbound chemotherapeutics) penetrate into the tumor but washed out within minutes, while dextran ∼30KdA (d=12nm) retain longer, but penetrate less due to the limited vascular permeability. MSV were efficiently Internalized by murine and human macrophages in vitro and by liver macrophages in vivo in the metastatic liver. MSV encapsulated Abraxane enabled an almost complete elimination of the metastatic loci, while Abraxane was not efficient. The initial therapy data suggest that MSV targeted macrophages in breast cancer liver metastasis enabling high concentrations of chemotherapeutic agents to accumulate in the tumor loci. This can be an efficient strategy for overcoming physical barriers in breast cancer liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2453. doi:1538-7445.AM2012-2453

New paramagnetic nanoconstructs (nCS) are demonstrated with superior MRI and thermal ablation properties. The nCSs are generated by loading Gd-DTPA and ultra-short gadonanotubes (GNTs) within the nanoporous matrix of systemically injectable silicon nanoporous particles (SiNPs) [1]. Gd-DTPA is a Gd-based contrast agent currently used in clinical practice. The GNTs consist of Gd3+-ion clusters encapsulated within carbon nanotube capsules, which are 20-80 nm in length and 1.3 nm in diameter. Nanoporous discoidal SiNPs are fabricated by combining optical lithography and electrochemical etching. Two different particle sizes (small 600×200 nm and large 1000×400 nm) and particle surface configurations (untargeted and RGD-4C targeted) are considered in this study. The nCSs are characterized in terms of (i) biodistribution in tumor bearing mice; (ii) MRI longitudinal relaxivity r1 and (iii) thermal ablation efficiency. The nCS accumulation in 6 organs (liver, spleen, heart, lungs, kidneys and brain) and in tumors is quantified by elemental silicon analysis through inductively coupled plasma-optical emission spectrometer. Organ accumulation is observed to highly dependent on particle shape and size. In tumors, the larger discoidal particles are observed to accumulate more than the smaller particles, with a percentage of injected dose per gram organ of about 5% and 1.5%, respectively. For RGD-4C targeted particles, the tumor accumulation percentages grow up to about 10% and 8% of the injected dose / gram tumor, respectively. For MRI applications, the nCSs exhibit a relaxivity enhancement up to 3-4 times the values of the original Gd-based imaging agent loaded (either Gd-DTPA or GNT). In particular, for the GNT loaded SiNPs, longitudinal relaxivities up to 160 mM-1s-1 per Gd3+-ions are measured at 1.5T, which are two orders of magnitude higher than for clinically available MRI agents (r1 = 4 mM-1s-1 per Gd3+-ions at 1.5T). Eventually, the nCS thermal ablating properties are characterized upon excitation through externally applied electromagnetic fields. High quality-factor resonators (0.5-300 MHz) with separated radio frequency -electric (up to 200 V/m) and -magnetic (up to 5 mT) fields are used for non-invasive nCS stimulation and fiber optic temperature sensors are used to map the associated temperature increase. The nCSs have shown both magnetic and dielectric rf losses and significant heating at 120 MHz. Mechanisms and efficiency of heating are discussed in terms of both magnetic and electric fields excitation, the concentration of nCSs, loading of GNTs into nCSs, frequency and power of the external generator.The multifunctionality and superior biodistribution performance of these nCSs could be effectively used in cancer imaging and treatment through alternative physical-based approaches. [1] Ananta JS,…, Decuzzi P. Nat Nanotechnol. 2010;5:815-21 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5299. doi:10.1158/1538-7445.AM2011-5299

The clinical applicability of antibodies and plasmonic nanosensors as topically applied, molecule-specific optical diagnostic agents for noninvasive early detection of cancer and precancer is severely limited by our inability to efficiently deliver macromolecules and nanoparticles through mucosal tissues. We have developed an imidazole-functionalized conjugate of the polysaccharide chitosan (chitosan-IAA) to enhance topical delivery of contrast agents, ranging from small molecules and antibodies to gold nanoparticles up to 44 nm in average diameter. Contrast agent uptake and localization in freshly resected mucosal tissues was monitored using confocal microscopy. Chitosan-IAA was found to reversibly enhance mucosal permeability in a rapid, reproducible manner, facilitating transepithelial delivery of optical contrast agents. Permeation enhancement occurred through an active process, resulting in the delivery of contrast agents via a paracellular or a combined paracellular/transcellular route depending on size. Coadministration of epidermal growth factor receptor-targeted antibodies with chitosan-IAA facilitated specific labeling and discrimination between paired normal and malignant human oral biopsies. Together, these data suggest that chitosan-IAA is a promising topical permeation enhancer for mucosal delivery of optical contrast agents.

Uniform delivery of optical contrast agents through mu- cosal tissue has proven a significant challenge. Topical permeation enhancers that have proven useful for skin demonstrate limited suc- cess in mucosal tissue. We sought to develop a topical permeation strategy capable of delivering tissue-impermeant molecular-specific contrast agents through mucosal epithelium in a uniform, controlled manner. We demonstrate that Triton-X100 can be utilized to deliver targeted and untargeted optical contrast agents through freshly ex- cised normal mucosal epithelium and epithelial cancer. Macromol- ecules up to 150 kDa in size were successfully delivered via transcel- lular and paracellular routes. The depth of Triton-mediated permeation was modulated by varying the treatment time and con- centration. Uniform epithelial penetration to a depth of 500 m was achieved in 1.5 h for molecules of 40 kDa or less. Larger optical probes required longer treatment times. Coadministration of molecular-specific contrast agents with Triton-X100 treatment facili- tated simultaneous labeling of biomarkers on the cell membrane, in the cytoplasm, and in the nucleus with high specificity. Together, these data suggest that Triton-X100 is a promising topical permeation enhancer for mucosal delivery of tissue-impermeant molecular- specific optical contrast agents. © 2009 Society of Photo-Optical Instrumentation