SF1: IFNβ production from macrophages by ELISA analysis; SF2: FMT imaging of mammary tumors ex vivo for spatial localization of nanoparticle; SF3: Confocal micrographs of DiI-nanoparticle fluorescence in mammary tumor tissue; SF4: Representative images of tumor masses 4 days post-treatment; SF5: Primary tumor bioluminescence and change in weight in 4T1 tumor-bearing mice; SF6: Confocal microscopy of a representative healthy lung section; SF7: Change in weight in B16F10 tumor-bearing mice

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon β, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, αvβ3 integrin and P-selectin that are commonly used to direct nanoparticles to tumors. Using the 4T1 mouse model, we assessed the microdistribution of the four NP variants in the tumor immune microenvironment in three different breast cancer landscapes, including primary tumor, early metastasis, and late metastasis. The different NP variants resulted in variable uptake by immune cell subsets depending on the organ and tumor stage. Among the NP variants, therapeutic studies indicated that the untargeted NPs and the integrin-targeting NPs exhibited a remarkable short- and long-term immune response and long-lasting antitumor effect.

The high mortality associated with glioblastoma multiforme (GBM) is attributed to its invasive nature, hypoxic core, resistant cell subpopulations and a highly immunosuppressive tumor microenvironment (TME). To support adaptive immune function and establish a more robust antitumor immune response, we boosted the local innate immune compartment of GBM using an immunostimulatory mesoporous silica nanoparticle, termed immuno-MSN. The immuno-MSN was specifically designed for systemic and proficient delivery of a potent innate immune agonist to dysfunctional antigen-presenting cells (APCs) in the brain TME. The cargo of the immuno-MSN was cyclic diguanylate monophosphate (cdGMP), a Stimulator of Interferon Gene (STING) agonist. Studies showed the immuno-MSN promoted the uptake of STING agonist by APCs in vitro and the subsequent release of the pro-inflammatory cytokine interferon β, 6-fold greater than free agonist. In an orthotopic GBM mouse model, systemically administered immuno-MSN particles were taken up by APCs in the near-perivascular regions of the brain tumor with striking efficiency. The immuno-MSNs facilitated the recruitment of dendritic cells and macrophages to the TME while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8+ T cell activity (2.5-fold) and delayed GBM tumor growth. We show that an engineered immunostimulatory nanoparticle can support pro-inflammatory innate immune function in GBM and subsequently augment current immunotherapeutic interventions and improve their therapeutic outcome.

The efficacy of immunotherapies is often limited by the immunosuppressive tumor microenvironment, which is populated with dysfunctional innate immune cells. To reprogram the tumor-resident innate immune cells, we developed immunostimulatory silica mesoporous nanoparticles (immuno-MSN). The cargo of immuno-MSN is a Stimulator of Interferon Gene (STING) agonist, which activates innate immune cells leading to production of interferon (IFN) β. By proficiently trafficking its cargo into immune cells, the immuno-MSN induced a 9-fold increase of IFN-β secretion compared to free agonist. While an external PEG shield has historically been used to protect nanoparticles from immune recognition, a PEGylated immunostimulatory nanoparticle needs to strike a balance between immune evasion to avoid off-site accumulation and uptake by target immune cells in tumors. Using the 4T1 mouse model of metastatic breast cancer and flow cytometry, it was determined that the degree of PEGylation significantly influenced the uptake of 'empty' MSNs by tumor-resident innate immune cells. This was not the case for the agonist-loaded immuno-MSN variants. It should be noted the surface charge of the 'empty' MSNs was positive rather than neutral for the agonist-loaded immuno-MSNs. However, even though the cellular uptake was similar at 24 h after injection for the three immuno-MSN variants, we observed a significant beneficial effect on the activation and expansion of APCs especially in lung metastasis using the lightly PEGylated immuno-MSN variant.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease, where even surgical resection and aggressive chemotherapy produce dismal outcomes. Immunotherapy is a promising alternative to conventional treatments, possessing the ability to elicit T cell-mediated killing of tumor cells and prevent disease recurrence. Immunotherapeutic approaches thus far have seen limited success in PDAC due to a poorly immunogenic and exceedingly immunosuppressive tumor microenvironment, which is enriched with dysfunctional and immunosuppressed antigen-presenting cells (APCs). We developed a highly potent immunostimulatory nanoparticle (immuno-NP) to activate and expand APCs in the tumor and induce local secretion of interferon β (IFNβ), which is a pro-inflammatory cytokine that plays a major role in APC recruitment. The effectiveness of the immuno-NP stems from its dual cargo of two synergistic immune modulators consisting of an agonist of the stimulator of interferon genes (STING) pathway and an agonist of the Toll-like receptor 4 (TLR4) pathway. We show the functional synergy of the dual-agonist cargo can be tweaked by adjusting the ratio of the two agonists loaded in the immuno-NP, leading to an increase in IFNβ production (11-fold) compared to any single agonist immuno-NP variant. Using the orthotopic murine Panc02 model of PDAC, we show that systemic administration allowed immuno-NPs to deposit into the perivascular regions of the tumor, which coincided with the APC-rich tumor areas leading to predominant uptake of immuno-NPs by APCs. The immuno-NPs were effectively taken up by a significant portion of dendritic cells in the tumor (>56%). This led to a significant expansion of APCs, resulting in an 11.5-fold increase of dendritic cells and infiltration of lymphocytes throughout the pancreatic tumor compared to untreated animals.

In situ forming implants (ISFIs) allow for a high initial intratumoral concentration and sustained release of the chemotherapeutic. However, clinical translation is impeded primarily due to limited drug penetration from the tumor/boundary interface and poor intratumoral drug retention. Therapeutic ultrasound (TUS) has become a popular approach for improving drug penetration of transdermal devices and increasing cellular uptake of nanoparticles. These effects are driven by the mechanical and thermal bioeffects associated with TUS. In this study, we characterize the released drug penetration, retention, and overall therapeutic response when exposing ISFI to the combination of the mechanical and thermal effects of TUS (C-TUS). ISFIs were intratumorally injected into subcutaneous murine tumors then exposed to C-TUS (exposure: 5 min, duty factor: 0.33, frequency: 3 MHz, intensity: 2.2 W/cm(2), pulse duration: 2 ms, pulse repetition frequency: 165 Hz, effective radiating area: 5 cm(2), energy delivered: 896 J, time average intensity: 0.88 W/cm(2)). Tumors treated with the combination of ISFI + C-TUS demonstrated a 2.5-fold increase in maximum drug penetration and a 3-fold increase in drug retention at 5- and 8-days post-injection, respectively, compared to ISFIs without TUS exposure. These improvements in drug penetration and retention translated into an enhanced therapeutic response. Mice treated with ISFI + C-TUS showed a 62.6% reduction in tumor progression, a 50.0% increase in median survival time, and a 26.6% increase in necrotic percentage compared to ISFIs without TUS exposure. Combining intratumoral ISFIs with TUS may be beneficial for addressing some long-standing challenges with local drug delivery in cancer treatment and may serve as a viable noninvasive method to improve the poor clinical success of local drug delivery systems.

Glioblastomas are highly lethal cancers defined by resistance to conventional therapies and rapid recurrence. While new brain tumor cell-specific drugs are continuously becoming available, efficient drug delivery to brain tumors remains a limiting factor. We developed a multicomponent nanoparticle, consisting of an iron oxide core and a mesoporous silica shell that can effectively deliver drugs across the blood-brain barrier into glioma cells. When exposed to alternating low-power radiofrequency (RF) fields, the nanoparticle's mechanical tumbling releases the entrapped drug molecules from the pores of the silica shell. After directing the nanoparticle to target the near-perivascular regions and altered endothelium of the brain tumor via fibronectin-targeting ligands, rapid drug release from the nanoparticles is triggered by RF facilitating wide distribution of drug delivery across the blood-brain tumor interface.

Deposition of nanoparticles to tumors often can be enhanced by targeting receptors overexpressed in a tumor. However, a tumor may exhibit a finite number of a biomarker that is accessible and targetable by nanoparticles, limiting the available landing spots. To explore this, we selected two different biomarkers that effectively home nanoparticles in brain tumors. Specifically, we used either an αvβ3 integrin-targeting peptide or a fibronectin-targeting peptide as a ligand on nanoparticles termed RGD-NP and CREKA-NP, respectively. In mouse models of glioblastoma multiforme, we systemically injected the nanoparticles loaded with a cytotoxic drug at different doses ranging from 2 to 8 mg/kg drug. The upper dose threshold of RGD-NP is ∼2 mg/kg. CREKA-NP reached its upper dose threshold at 5 mg/kg. For both targeted nanoparticle variants, higher dose did not ensure higher intratumoral drug levels, but it contributed to elevated off-target deposition and potentially greater toxicity. A cocktail combining RGD-NP and CREKA-NP was then administered at a dose corresponding to the upper dose threshold for each formulation resulting in a 3-fold higher intratumoral deposition than the individual formulations. The combination of the two different targeting schemes at the appropriate dose for each nanoparticle variant facilitated remarkable increase in intratumoral drug levels that was not achievable by a sole targeting nanoparticle alone.

Glioblastoma multiforme (GBM) remains highly lethal. This partially stems from the presence of brain tumor initiating cells (BTICs), a highly plastic cellular subpopulation that is resistant to current therapies. In addition to resistance, the blood–brain barrier limits the penetration of most drugs into GBMs. To effectively deliver a BTIC‐specific inhibitor to brain tumors, a multicomponent nanoparticle, termed Fe@MSN, which contains a mesoporous silica shell and an iron oxide core, is developed. Fibronectin‐targeting ligands direct the nanoparticle to the near‐perivascular areas of GBM. After Fe@MSN particles are deposited in the tumor, an external low‐power radiofrequency (RF) field triggers rapid drug release due to mechanical tumbling of the particle resulting in penetration of high amounts of drug across the blood–brain tumor interface and widespread drug delivery into the GBM. The nanoparticle is loaded with the drug 1400W, which is a potent inhibitor of the inducible nitric oxide synthase (iNOS). It is shown that iNOS is preferentially expressed in BTICs and is required for their maintenance. Using the 1400W‐loaded Fe@MSN and RF‐triggered release, in vivo studies indicate that the treatment disrupts the BTIC population in hypoxic niches, suppresses tumor growth and significantly increases survival in BTIC‐derived GBM xenografts.

Abstract Effective cancer immunotherapy depends on the robust activation of tumor-specific antigen-presenting cells (APC). Immune agonists encapsulated within nanoparticles (NP) can be delivered to tumor sites to generate powerful antitumor immune responses with minimal off-target dissemination. Systemic delivery enables widespread access to the microvasculature and draining to the APC-rich perivasculature. We developed an immuno-nanoparticle (immuno-NP) coloaded with cyclic diguanylate monophosphate, an agonist of the stimulator of interferon genes pathway, and monophosphoryl lipid A, and a Toll-like receptor 4 agonist, which synergize to produce high levels of type I IFNβ. Using a murine model of metastatic triple-negative breast cancer, systemic delivery of these immuno-NPs resulted in significant therapeutic outcomes due to extensive upregulation of APCs and natural killer cells in the blood and tumor compared with control treatments. These results indicate that NPs can facilitate systemic delivery of multiple immune-potentiating cargoes for effective APC-driven local and systemic antitumor immunity. Significance: Systemic administration of an immuno-nanoparticle in a murine breast tumor model drives a robust tumor site–specific APC response by delivering two synergistic immune-potentiating molecules, highlighting the potential of nanoparticles for immunotherapy.

Issues with limited intratumoral drug penetration and heterogeneous drug distribution continue to impede the therapeutic efficacy of nanomedicine-based delivery systems. Ultrasound (US)-enhanced drug delivery has emerged as one effective means of overcoming these challenges. Acoustic cavitation in the presence of nanoparticles has shown to increase the cellular uptake and distribution of chemotherapeutic agents in vivo. In this study, we investigated the potential of a drug-loaded echogenic nanoscale bubbles in combination with low frequency (3 MHz), high energy (2 W/cm2) US for antitumor therapy. The doxorubicin-loaded nanobubbles (Dox-NBs) stabilized with an interpenetrating polymer mesh were 171.5 ± 20.9 nm in diameter. When used in combination with therapeutic US, Dox-NBs combined with free drug showed significantly higher (*p < 0.05) intracellular uptake and therapeutic efficacy compared with free drug. When injected intravenously in vivo, Dox-NBs + therapeutic US showed significantly higher (*p < 0.05) accumulation and better distribution of Dox in tumors when compared with free drug. This strategy provides an effective and simple method to increase the local dose and distribution of otherwise systemically toxic chemotherapeutic agents for cancer therapies.

Local drug delivery systems, such as in situ forming implants (ISFIs), allow for sustained elevated drug concentration directly at the tumor site. However, these systems have been challenging to translate into clinical practice due to poor drug penetration through the implant/tumor boundary. Ultrasound (US) has emerged as a popular approach to enhance drug release and cellular drug uptake from nanoparticles, but little work has been done with its use in combination with ISFIs. In this study, ISFIs were intratumorally injected and treated with therapeutic ultrasound (TUS). A significant (p <0.05) increase was seen in Doxorubicin (Dox) distribution with ISFIs treated with TUS as compared to ISFIs not treated with TUS. Additionally, the combination of Dox ISFIs with TUS showed a significant (p <0.01) reduction in tumor growth at 20 days, compared to all other treatment groups. While the mechanism of increased drug distribution and enhanced therapeutic efficacy is not yet clear, hyperthermia was seen with all groups that involved application of TUS for these US exposure parameters. This could be attributed to elevated drug penetration and synergy with the chemotherapy, but additional experiments need to confirm this hypothesis. This study demonstrates, for the first time, US-enhanced drug distribution from ISFIs. The combination of US with local chemotherapy could eventually facilitate translation of local drug delivery systems into clinical practice.

Sonodynamic therapy (SDT) has become a promising noninvasive approach for cancer therapy. The treatment exploits the ability of particular molecules (i.e., porphyrins) to be excited by ultrasound and produce reactive oxygen species (ROS) during their decay process. These reactive species, in turn, result in cell death. To capitalize on the real-time visualization and on-demand delivery of ultrasound contrast agents, this study aims to combine porphyrins with nanobubbles (NBs) to obtain an ultrasound-activated theranostic agent that exploits the SDT activity in vitro. Two porphyrin classes, exposing different hydrophobic side chains, were synthesized. NB size and encapsulation efficiency were markedly dependent on the porphyrin structure. The combination of these porphyrin and NBs resulted in a significant reduction in cell viability upon sonication in pilot studies performed on the LS 174T colorectal cancer cell line.

Event Abstract Back to Event Beyond the EPR effect: targeting of nanoparticles to pediatric brain tumors Elizabeth Doolittle1, 2, 3, Peter Bielecki1, 2, 3, Amy Goldberg1 and Efstathios Karathanasis1, 2, 3 1 Case Western Reserve University, Biomedical Engineering, United States 2 Case Western Reserve University, Radiology, United States 3 Case Western Reserve University, Case Comprehensive Cancer Center, United States Introduction: In cancer applications, nanoparticles should selectively deposit to tumor areas due to the enhanced permeability and retention (EPR) effect. However, recent reports generated controversy over the benefits of EPR. Further, in the case of brain tumors, while the blood-brain barrier is compromised, tumor-associated blood vessels are not as leaky as in other tumor types. We previously showed vascular targeting nanoparticles lead to successful imaging of breast tumor micrometastatic sites (Figure 1) [1],[2] suggesting an approach appropriate for tumor types in which EPR alone is ineffective. Here, we investigated nanoparticles in murine models of adult and pediatric glioblastoma multiforme for targeted particle delivery for imaging and therapy. Materials and Methods: Experiments used invasive glioma orthotopic murine models: an established murine glioma (CNS-1) and a patient derived pediatric brain tumor (SJ-GBM2). Biomarkers used for targeting were validated using tumor section immunohistochemistry. Various nanoparticles (liposomes, gold or iron oxide) with surface PEG-amines were conjugated with selected biomarker ligands and imaging probes for particle detection. Brains were inoculated with GFP-positive CNS-1 or SJ-GBM2 cells. Particles were intravenously injected and animals were sacrificed 8-14 days after tumor inoculation. Organs were analyzed using 2D and 3D fluorescence imaging. Histological analyses validated intratumoral deposition of particles. Results and Discussion: Targeting ligands were based on high affinity peptides against known receptors overexpressed by the brain tumor’s vascular bed (integrins, VEGFR2). Dynamic light scattering and TEM characterized particle size. Surface ligands added at varying surface densities (1000-2500 ligands per particle) were confirmed by direct protein assay. Animal studies showed patterns of nanoparticle deposition dependent on targeting strategy (vascular vs deep tissue). Vascular targeting was more effective than deep tissue targeting in most occasions, since deep tissue targeting requires prerequisite EPR. In particular, histological analyses showed that while EPR-driven deposition resulted in accumulation in cancerous sites, vascular targeting yielded higher, more consistent capture of the majority of tumor sites. Further, size of the nanoparticles and resulting multivalent avidity makes nanoparticles ideal to vascular targeting. In a recent study, vascular targeting of a gold nanoparticle radiotracer led to 5.2-fold higher deposition in lung metastases than the equivalent small molecule analogue. Figure 1 is an example of the result of vascular targeting. The left image gamma scintigraphy shows radiolabeled nanoparticle accumulation corresponding to GFP positive tumor cells shown in the right image[1]. Conclusion: When one considers the targeted tumor microenvironment’s biophysical and biochemical uniqueness, rationale design of nanoparticles and targeting strategy can result in enhancements. This work was supported by grants from the National Cancer Institute (U01CA198892, R01CA177716) and the Prayers from Maria Children's Glioma Cancer Foundation (E.K.).; E.D. and P.B were supported by the NIH Interdisciplinary Biomedical Imaging Training Program (5T32EB007509).References:[1] Doolittle, E. et al. ACS Nano 9, 8012 (2015).[2] Peiris, P. M. et al. J Pharm Sci 104, 2600 (2015). Keywords: Molecular Imaging, in vivo, nanoparticle, targeting delivery Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Cell-targeting biomaterials in theranostic delivery Citation: Doolittle E, Bielecki P, Goldberg A and Karathanasis E (2016). Beyond the EPR effect: targeting of nanoparticles to pediatric brain tumors. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00545 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Elizabeth Doolittle Peter Bielecki Amy Goldberg Efstathios Karathanasis Google Elizabeth Doolittle Peter Bielecki Amy Goldberg Efstathios Karathanasis Google Scholar Elizabeth Doolittle Peter Bielecki Amy Goldberg Efstathios Karathanasis PubMed Elizabeth Doolittle Peter Bielecki Amy Goldberg Efstathios Karathanasis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Event Abstract Back to Event Precise targeting of pediatric brain tumors using chain-like nanoparticles Peter Bielecki1, 2, 3, Elizabeth Doolittle1, 2, 3, Pubudu Peiris1, 2, 3 and Efstathios Karathanasis1, 2, 3 1 Case Western Reserve University, Department of Biomedical Engineering, United States 2 Case Western Reserve University, Department of Radiology, United States 3 Case Western Reserve University, Case Comprehensive Cancer Center, United States Introduction: Aggressive pediatric brain tumors are terminal upon diagnosis, yet no new therapeutic protocols have been developed in over 30 years. Glioblastoma multiforme is considered recalcitrant to current surgical and local radiotherapeutic approaches, while systemic chemotherapeutic approaches are impeded by the blood-tumor barrier. However, these invasive brain tumors upregulate the αv­­­β3 integrin at tumor vasculature to promote angiogenesis, which differ from the surrounding tissue. Thus, the αv­­­β3 integrin provides a unique opportunity for vascular targeting of a nanoparticle imaging agent to glioma sites. We fabricated an integrin-targeted nanoparticle composed of multiple iron oxide (IO) nanospheres chemically assembled into a linear nanochain [1],[2]. Compared to small molecules or spherical nanoparticles, we show that the flexible, oblate shape of the integrin-targeted nanochains can facilitate superior and accurate targeting of the brain tumor’s vascular bed due to geometrically enhanced multivalent docking onto blood vessels associated with brain tumors. Materials and Methods: Using well-established methods [3], nanochains were synthesized and a cyclic RGD peptide was attached for targeting. In vivo efficacy studies were conducted in mouse orthotopic models of pediatric glioblastoma. Brain tumors were induced by intracranial injection of two GFP-expressing cell lines (SJ-GBM2 and CHLA 200) into antithymic mice. Following intravenous injection of nanochains, in vivo fluorescent imaging was performed to non-invasively and quantitatively monitor the time-course of tumor deposition of the particles. Initially, we compared the targeted nanochains to targeted nanospheres and their non-targeting variants in their ability to target pediatric brain tumors. To confirm the findings of in vivo imaging, post mortem histological analyses were performed. Results and Discussion: The shape and prolonged blood residence time of the nanochain resulted in increased interactions with the vascular bed of the primary site of brain tumors as well as their infiltrating edges. As expected, the non-targeted spheres or nanochains slowly accumulate into the tumor via the EPR effect. However, targeted nanochains displayed at least 5-fold higher tumor deposition than their non-targeting variant. Compared to spherical nanoparticles, chain-like particles resulted in superior targeting of αvβ3 integrins due to geometrically enhanced multivalent docking. Vascular targeting via the RGD peptide resulted in >4% of the administered nanochains attached to the tumor within ~30 min after injection. Using a fluorescence imaging system, ex vivo imaging confirmed nanoparticle accumulation at the glioma sites. Similar to optical imaging, MR imaging accurately detected the cancer. Conclusions: Our data indicate that a vascular targeting strategy can selectively target the nanochain particles to pediatric glioblastoma multiforme resulting in tumor detection by optical imaging or MRI. This work was supported by grants from the National Cancer Institute (U01CA198892, R01CA177716) and the Prayers from Maria Children's Glioma Cancer Foundation (E.K.).; E.D. and P.B were supported by the NIH Interdisciplinary Biomedical Imaging Training Program (5T32EB007509).References:[1] Peiris, P. M. et al. ACS Nano 6, 8783 (2012).[2] Peiris, P. M. et al. Cancer Research 75, 1356 (2015).[3] Peiris, P. M. et al. PLoS One 6, e15927 (2011). Keywords: Molecular Imaging, in vivo, nanoparticle, targeting delivery Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Cell-targeting biomaterials in theranostic delivery Citation: Bielecki P, Doolittle E, Peiris P and Karathanasis E (2016). Precise targeting of pediatric brain tumors using chain-like nanoparticles. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02625 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Peter Bielecki Elizabeth Doolittle Pubudu Peiris Efstathios Karathanasis Google Peter Bielecki Elizabeth Doolittle Pubudu Peiris Efstathios Karathanasis Google Scholar Peter Bielecki Elizabeth Doolittle Pubudu Peiris Efstathios Karathanasis PubMed Peter Bielecki Elizabeth Doolittle Pubudu Peiris Efstathios Karathanasis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

.............................................................................................................................. ii Acknowledgments.............................................................................................................. iii Vita ..................................................................................................................................... iv List of Figures .................................................................................................................... vi Chapter 1: Background ...................................................................................................... 1 Chapter 2: Project Introduction ........................................................................................... 2 Chapter 3: Materials and Methods ..................................................................................... 4 Core-Shell Microfluidic Device Fabrication .................................................................. 4 Experimental Procedure ................................................................................................. 5 Chapter 4: Results ............................................................................................................... 6 Core-Shell Microcapsule Confirmation .......................................................................... 6 Core-Shell Microcapsules of Varying Stiffness ............................................................... 7 Control Culture of HEPM Stem Cells in 3D Collagen Matrix ....................................... 8 Encapsulation of HEPM Stem Cells in 3D Collagen Cores ........................................... 9 Chapter 5: Discussion ....................................................................................................... 11 References ......................................................................................................................... 14

A novel core-shell microcapsule system is developed in this study to mimic the miniaturized 3D architecture of pre-hatching embryos with an aqueous liquid-like core of embryonic cells and a hydrogel-shell of zona pellucida. This is done by microfabricating a non-planar microfluidic flow-focusing device that enables one-step generation of microcapsules with an alginate hydrogel shell and an aqueous liquid core of cells from two aqueous fluids. Mouse embryonic stem (ES) cells encapsulated in the liquid core are found to survive well (>92%). Moreover, ~20 ES cells in the core can proliferate to form a single ES cell aggregate in each microcapsule within 7 days while at least a few hundred cells are usually needed by the commonly used hanging-drop method to form an embryoid body (EB) in each hanging drop. Quantitative RT-PCR analyses show significantly higher expression of pluripotency marker genes in the 3D aggregated ES cells compared to the cells under 2D culture. The aggregated ES cells can be efficiently differentiated into beating cardiomyocytes using a small molecule (cardiogenol C) without complex combination of multiple growth factors. Taken together, the novel 3D microfluidic and pre-hatching embryo-like microcapsule systems are of importance to facilitate in vitro culture of pluripotent stem cells for their ever-increasing use in modern cell-based medicine.