Rachael W. Sirianni

Intravenous delivery of camptothecin-loaded PLGA nanoparticles for the treatment of intracranial glioma

Effective treatment of glioblastoma multiforme remains a major clinical challenge, due in part to the difficulty of delivering chemotherapeutics across the blood-brain barrier. Systemically administered drugs are often poorly bioavailable in the brain, and drug efficacy within the central nervous system can be limited by peripheral toxicity. Here, we investigate the ability of systemically administered poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) to deliver hydrophobic payloads to intracranial glioma. Hydrophobic payload encapsulated within PLGA NPs accumulated at ∼10× higher levels in tumor compared to healthy brain. Tolerability of the chemotherapeutic camptothecin (CPT) was improved by encapsulation, enabling safe administration of up to 20mg/kg drug when encapsulated within NPs. Immunohistochemistry staining for γ-H2AFX, a marker for double-strand breaks, demonstrated higher levels of drug activity in tumors treated with CPT-loaded NPs compared to free drug. CPT-loaded NPs were effective in slowing the growth of intracranial GL261 tumors in immune competent C57 albino mice, providing a significant survival benefit compared to mice receiving saline, free CPT or low dose CPT NPs (median survival of 36.5 days compared to 28, 32, 33.5 days respectively). In sum, these data demonstrate the feasibility of treating intracranial glioma with systemically administered nanoparticles loaded with the otherwise ineffective chemotherapeutic CPT.

PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering

UNLABELLED Injectable biomaterials offer a non-invasive approach to deliver cells into the myocardial infarct region to maintain a high level of cell retention and viability and initiate the regeneration process. However, previously developed injectable matrices often suffer from low bioactivity or poor mechanical properties. To address this need, we introduced a biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with excellent bioactivity as well as mechanical robustness for cardiac tissue engineering. A unique feature of our work was that we performed extensive in vitro biological analyses to assess the functionalities of cardiomyocytes (CMs) alone and in co-culture with cardiac fibroblasts (CFs) (2:1 ratio) within the hydrogel matrix. The synthesized hydrogel exhibited viscoelastic behavior (storage modulus: 1260 Pa) and necessary water content (75%) to properly accommodate the cardiac cells. The encapsulated cells demonstrated a high level of cell survival (90% for co-culture condition, day 7) and spreading throughout the hydrogel matrix in both culture conditions. A dense network of stained F-actin fibers (∼ 6 × 10(4) μm(2) area coverage, co-culture condition) illustrated the formation of an intact and three dimensional (3D) cell-embedded matrix. Furthermore, immunostaining and gene expression analyses revealed mature phenotypic characteristics of cardiac cells. Notably, the co-culture group exhibited superior structural organization and cell-cell coupling, as well as beating behavior (average ∼ 45 beats per min, co-culture condition, day 7). The outcome of this study is envisioned to open a new avenue for extensive in vitro characterization of injectable matrices embedded with 3D mono- and co-culture of cardiac cells prior to in vivo experiments. STATEMENT OF SIGNIFICANCE In this work, we synthesized a new class of biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with suitable bioactivity and mechanical properties for cardiac tissue engineering. A significant aspect of our work was that we performed extensive in vitro biological analyses to assess the functionality of cardiomyocytes alone and in co-culture with cardiac fibroblasts encapsulated within the 3D hydrogel matrix.

A critical evaluation of drug delivery from ligand modified nanoparticles: Confounding small molecule distribution and efficacy in the central nervous system

In this work, we sought to test how surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with peptide ligand alters the brain specific delivery of encapsulated molecules. For biodistribution studies, nanoparticles modified with rabies virus glycoprotein (RVG29) were loaded with small molecule drug surrogates and administered to healthy mice by lateral tail vein injection. Mice were perfused 2h after injection and major anatomical regions of the CNS were dissected (striatum, midbrain, cerebellum, hippocampus, cortex, olfactory bulb, brainstem, and cervical, thoracic, lumbar and sacral spinal cord). For functional studies, surface modified nanoparticles were loaded with the chemotherapeutic camptothecin (CPT) and administered to mice bearing intracranial GL261-Luc2 gliomas. Outcome measures included tumor growth, as measured by bioluminescent imaging, and median survival time. We observed that small molecule delivery from PLGA nanoparticles varied by as much as 150% for different tissue regions within the CNS. These differences were directly correlated to regional differences in cerebral blood volume. Although the presence of RVG29 enhanced apparent brain delivery for multiple small molecule payloads, we observed minimal evidence for targeting to muscle or spinal cord, which are the known sites for rabies virus entry into the CNS, and enhancements in brain delivery were not prolonged due to an apparent aqueous instability of the RVG29 ligand. Furthermore, we have identified concerning differences in apparent delivery kinetics as measured by different payloads: nanoparticle encapsulated DiR was observed to accumulate in the brain, whereas encapsulated Nile red was rapidly cleared. Although systemically administered CPT loaded nanoparticles slowed the growth of orthotopic brain tumors to prolong survival, the presence of RVG29 did not enhance therapeutic efficacy compared to control nanoparticles. These data are consistent with a model of delivery of hydrophobic small molecules to the brain that does not rely on internalization of polymer nanoparticles in target tissue. We discuss an important risk for discordance between biodistribution, as typically measured by drug surrogate, and therapeutic outcome, as determined by clinically relevant measurement of drug function in a disease model. These results pose critical considerations for the methods used to design and evaluate targeted drug delivery systems in vivo.

Hyaluronic acid-laminin hydrogels increase neural stem cell transplant retention and migratory response to SDF-1α

The chemokine SDF-1α plays a critical role in mediating stem cell response to injury and disease and has specifically been shown to mobilize neural progenitor/stem cells (NPSCs) towards sites of neural injury. Current neural transplant paradigms within the brain suffer from low rates of retention and engraftment after injury. Therefore, increasing transplant sensitivity to injury-induced SDF-1α represents a method for increasing neural transplant efficacy. Previously, we have reported on a hyaluronic acid-laminin based hydrogel (HA-Lm gel) that increases NPSC expression of SDF-1α receptor, CXCR4, and subsequently, NPSC chemotactic migration towards a source of SDF-1α in vitro. The study presented here investigates the capacity of the HA-Lm gel to promote NPSC response to exogenous SDF-1α in vivo. We observed the HA-Lm gel to significantly increase NPSC transplant retention and migration in response to SDF-1α in a manner critically dependent on signaling via the SDF-1α-CXCR4 axis. This work lays the foundation for development of a more effective cell therapy for neural injury, but also has broader implications in the fields of tissue engineering and regenerative medicine given the essential roles of SDF-1α across injury and disease states.

Temperature responsive hydrogels enable transient three-dimensional tumor cultures via rapid cell recovery.

Recovery of live cells from three-dimensional (3D) culture would improve analysis of cell behaviors in tissue engineered microenvironments. In this work, we developed a temperature responsive hydrogel to enable transient 3D culture of human glioblastoma (GBM) cells. N-isopropylacrylamide was copolymerized with hydrophilic grafts and functionalized with the cell adhesion peptide RGD to yield the novel copolymer poly(N-isopropylacrylamide-co-Jeffamine(®) M-1000 acrylamide-co-hydroxyethylmethacrylate-RGD), or PNJ-RGD. This copolymer reversibly gels in aqueous solutions when heated under normal cell culture conditions (37°C). Moreover, these gels redissolve within 70 s when cooled to room temperature without the addition of any agents to degrade the synthetic scaffold, thereby enabling rapid recollection of viable cells after 3D culture. We tested the efficiency of cell recovery following extended 3D culture and were able to recover more than 50% of viable GBM cells after up to 7 days in culture. These data demonstrate the utility of physically crosslinked PNJ-RGD hydrogels as a platform for culture and recollection of cells in 3D.

Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo

&NA; Understanding of the mechanisms by which systemically administered nanoparticles achieve delivery across biological barriers remains incomplete, due in part to the challenge of tracking nanoparticle fate in the body. Here, we develop a new approach for “barcoding” nanoparticles composed of poly(lactic‐co‐glycolic acid) (PLGA) with bright, spectrally defined quantum dots (QDs) to enable direct, fluorescent detection of nanoparticle fate with subcellular resolution. We show that QD labeling does not affect major biophysical properties of nanoparticles or their interaction with cells and tissues. Live cell imaging enabled simultaneous visualization of the interaction of control and targeted nanoparticles with bEnd.3 cells in a flow chamber, providing direct evidence that surface modification of nanoparticles with the cell‐penetrating peptide TAT increases their biophysical association with cell surfaces over very short time periods under convective current. We next developed this technique for quantitative biodistribution analysis in vivo. These studies demonstrate that nanoparticle surface modification with the cell penetrating peptide TAT facilitates brain‐specific delivery that is restricted to brain vasculature. Although nanoparticle entry into the healthy brain parenchyma is minimal, with no evidence for movement of nanoparticles across the blood‐brain barrier (BBB), we observed that nanoparticles are able to enter to the central nervous system (CNS) through regions of altered BBB permeability – for example, into circumventricular organs in the brain or leaky vasculature of late‐stage intracranial tumors. In sum, these data demonstrate a new, multispectral approach for barcoding PLGA, which enables simultaneous, quantitative analysis of the fate of multiple nanoparticle formulations in vivo. Graphical abstract TEM image (top left panel) of PLGA nanoparticle covalently linked to quantum dots. These nanoparticles can be used to study nanoparticle fate in cells, in healthy tissue (liver) and in diseased tissue (intracranial tumor core) with high spatial resolution and multiplex capability. Figure. No caption available.

Tailoring Sub-Micron PLGA Particle Release Profiles via Centrifugal Fractioning.

Poly(D,L-lactic-co -glycolic) acid (PLGA)-based sub-micron particles are uniquely posed to overcome limitations of conventional drug delivery systems. However, tailoring cargo/payload release profiles from PLGA micro/nanoparticles typically requires optimization of the multi-parameter formulation, where small changes may cause drastic shifts in the resulting release profiles. In this study, we aimed to establish whether refining the average diameter of sub-micron particle populations after formulation alters protein release profiles. PLGA particles were first produced via double emulsion-solvent evaporation method to encapsulate bovine serum albumin. Particles were then subjected to centrifugal fractioning protocols varying in both spin time and force to determine encapsulation efficiency and release profile of differently sized populations that originated from a single batch. We found the average particle diameter was related to marked alterations in encapsulation efficiencies (range: 36.4-49.4%), burst release (range: 15.8-49.1%), and time for total cargo release (range: 38-78 days). Our data corroborate previous reports relating PLGA particle size with such release characteristics, however, this is the first study, to our knowledge, to directly compare particle population size while holding all formulation parameters constant. In summary, centrifugal fractioning to selectively control the population distribution of sub-micron PLGA particles represents a feasible tool to tailor release characteristics.

CRMP2 Phosphorylation Drives Glioblastoma Cell Proliferation.

Glioblastoma (GBM) is an aggressive primary brain tumor. The rapid growth and the privileged provenance of the tumor within the brain contribute to its aggressivity and poor therapeutic targeting. A poor prognostic factor in glioblastoma is the deletion or mutation of the Nf1 gene. This gene codes for the protein neurofibromin, a tumor suppressor gene that is known to interact with the collapsin response mediator protein 2 (CRMP2). CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 have recently been implicated in cancer progression. The CRMP2-neurofibromin interaction protects CRMP2 from its phosphorylation by cyclin-dependent kinase 5 (Cdk5), an event linked to cancer progression. In three human glioblastoma cell lines (GL15, A172, and U87), we observed an inverse correlation between neurofibromin expression and CRMP2 phosphorylation levels. Glioblastoma cell proliferation was dependent on CRMP2 expression and phosphorylation by Cdk5 and glycogen synthase kinase 3 beta (GSK3β). The CRMP2 phosphorylation inhibitor (S)-lacosamide reduces, in a concentration-dependent manner, glioblastoma cell proliferation and induced apoptosis in all three GBM cell lines tested. Since (S)-lacosamide is bioavailable in the brain, we tested its utility in an in vivo orthotopic model of GBM using GL261-LucNeo glioma cells. (S)-lacosamide decreased tumor size, as measured via in vivo bioluminescence imaging, by ~54% compared to vehicle control. Our results introduce CRMP2 expression and phosphorylation as a novel player in GBM proliferation and survival, which is enhanced by loss of Nf1.

Modeling Microenvironmental Regulation of Glioblastoma Stem Cells: A Biomaterials Perspective

Following diagnosis of a glioblastoma (GBM) brain tumor, surgical resection, chemotherapy and radiation together yield a median patient survival of only 15 months. Importantly, standard treatments fail to address the dynamic regulation of the brain tumor microenvironment that actively supports tumor progression and treatment resistance. It is becoming increasingly recognized that specialized niches within the tumor microenvironment maintain a population of highly malignant glioblastoma stem-like cells (GSCs). GSCs are resistant to traditional chemotherapy and radiation therapy, suggesting that they may be responsible for the near universal rates of tumor recurrence and associated morbidity in GBM. Thus, disrupting microenvironmental support for GSCs could be critical to developing more effective GBM therapies. Three-dimensional (3D) culture models of the tumor microenvironment are powerful tools for identifying key biochemical and biophysical inputs that impact malignant behaviors. Such systems have been used effectively to identify conditions that regulate GSC proliferation, invasion, stem-specific phenotypes, and treatment resistance. Considering the significant role that GSC microenvironments play in regulating this tumorigenic sub-population, these models may be essential for uncovering mechanisms that limit GSCs malignancy.

pH driven precipitation of quisinostat onto PLA-PEG nanoparticles enables treatment of intracranial glioblastoma

Histone deacetylases (HDACs) are known to be key enzymes in cancer development and progression through their modulation of chromatin structure and the expression and post-translational modification of numerous proteins. Aggressive dedifferentiated tumors, like glioblastoma, frequently overexpress HDACs, while HDAC inhibition can lead to cell cycle arrest, promote cellular differentiation and induce apoptosis. Although multiple HDAC inhibitors, such as quisinostat, are of interest in oncology due to their potent in vitro efficacy, their failure in the clinic as monotherapies against solid tumors has been attributed to poor delivery. Thus, we were motivated to develop quisinostat loaded poly(D,L-lactide)-b-methoxy poly(ethylene glycol) nanoparticles (NPs) to test their ability to treat orthotopic glioblastoma. In developing our NP formulation, we identified a novel, pH-driven approach for achieving over 9% (w/w) quisinostat loading. We show quisinostat-loaded NPs maintain drug potency in vitro and effectively slow tumor growth in vivo, leading to a prolonged survival compared to control mice.