Highly stable, ligand-clustered “patchy” micelle nanocarriers for systemic tumor targeting

A novel linear-dendritic block copolymer has been synthesized and evaluated for targeted delivery. The use of the dendron as the micellar exterior block in this architecture allows the presentation of a relatively small quantity of ligands in clusters for enhanced targeting, thus maintaining a long circulation time of these “patchy” micelles. The polypeptide linear hydrophobic block drives formation of micelles that carry core-loaded drugs, and their unique design gives them extremely high stability in vivo. We have found that these systems lead to extended time periods of increased accumulation in the tumor (up to 5 days) compared with nontargeted vehicles. We also demonstrate a fourfold increase in efficacy of paclitaxel when delivered in the targeted nanoparticle systems, while significantly decreasing in vivo toxicity of the chemotherapy treatment.National Institute for Biomedical Imaging and Bioengineering (U.S.)National Cancer Institute (U.S.) (R01EB008082-01A2

Self-assembled RNA interference microsponges for efficient siRNA delivery

The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles1, 2, cationic complexes3, 4, inorganic nanoparticles5, 6, 7, 8, RNA nanoparticles9, 10 and dendrimers11. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic1, 12, 13. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.National Institutes of Health (U.S.) (NIH) NIBIB Grant R01-EB008082)United States. American Recovery and Reinvestment Act of 2009 ((ARRA) grant)National Science Foundation (U.S.) (Division of Materials Research Polymers Program grant #0705234)David H. Koch Institute for Integrative Cancer Research at MIT (Nanotechnology grant

Hydrogen-bonded multilayer of pH-responsive polymeric micelles with tannic acid for surface drug delivery

We report the design of a platform for the delivery of hydrophobic drugs conjugated to block copolymer micelles via pH-responsive linkage that are assembled within hydrogen-bonded polymer multilayer thin films.close465

Osteotropic Therapy via Targeted Layer-by-Layer Nanoparticles

Current treatment options for debilitating bone diseases such as osteosarcoma, osteoporosis, and bone metastatic cancer are suboptimal and have low efficacy. New treatment options for these pathologies require targeted therapy that maximizes exposure to the diseased tissue and minimizes off-target side effects. This work investigates an approach for generating functional and targeted drug carriers specifically for treating primary osteosarcoma, a disease in which recurrence is common and the cure rate has remained around 20%. This approach utilizes the modularity of Layer-by-Layer (LbL) assembly to generate tissue-specific drug carriers for systemic administration. This is accomplished via surface modification of drug-loaded nanoparticles with an aqueous polyelectrolyte, poly(acrylic acid) (PAA), side-chain functionalized with alendronate, a potent clinically used bisphosphonate. Nanoparticles coated with PAA-alendronate are observed to bind and internalize rapidly in human osteosarcoma 143B cells. Encapsulation of doxorubicin, a front-line chemotherapeutic, in an LbL-targeted liposome demonstrates potent toxicity in vitro. Active targeting of 143B xenografts in NCR nude mice with the LbL-targeted doxorubicin liposomes promotes enhanced, prolonged tumor accumulation and significantly improved efficacy. This report represents a tunable approach towards the synthesis of drug carriers, in which LbL enables surface modification of nanoparticles for tissue-specific targeting and treatment.National Institutes of Health (U.S.) (P30 CA14051 (NCI))National Institutes of Health (U.S.) (5 U54 CA151884–02 (CCNE))National Institutes of Health (U.S.) (R01 AG029601 (NIA))National Institutes of Health (U.S.) (R01 EB010246 (NIBIB))David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Swanson Biotechnology Center)National Science Foundation (U.S.) (Graduate Research Fellowship)National Health and Medical Research Council (Australia)Massachusetts Institute of Technology (David H. Koch (1962) Chair Professorship in Engineering

Ligand-clustered “patchy” nanoparticles for modulated cellular uptake and in vivo tumor targeting

Author Manuscript: 2012 August 05.A matter of presentation: The manner in which polyvalent ligands are presented to a cell—homogeneously or in spatially defined groupings on a nanoparticle surface—may play an important role in cellular uptake. This aspect is investigated for the first time using a linear dendritic polymer construct to pattern the surfaces of nanoparticles with variable-sized ligand clusters in different spatial arrangements.National Institutes of Health (U.S.) (NIH NIBIB Grant 5R01EB008082-02)MIT-Harvard Center of Cancer Nanotechnology ExcellenceNational Science Foundation (U.S.

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.National University of Singapore (Graduate School for Integrative Sciences and Engineering Program)Singapore-MIT Alliance (Singapore-MIT Alliance-3 graduate fellowship program)Singapore. National Research FoundationSingapore-MIT Alliance for Research and Technology (BioSystems and Micromechanics Interdisciplinary Research Group)Singapore. National Medical Research Council (NMRC/Clinician Scientist Award/012/2009

Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.United States. Dept. of Defense. Ovarian Cancer Research Program (Teal Innovator Award Grant OC120504)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32EB017614-01)National Science Foundation (U.S.). Graduate Research Fellowshi

The architecture and biological performance of drug-loaded LbL nanoparticles

Layer-by-Layer (LbL) nanoparticles are an emerging class of therapeutic carriers that afford precise control over key design parameters that facilitate improved drug and carrier pharmacokinetics, and enhanced molecular-targeting capabilities. This paper advances the development of these systems by establishing them as drug carriers, with the means to control drug release in a systemic environment and retard particle clearance from circulation, promoting improved biodistribution of the drug-containing system. Using dual-fluorescent tracking in vivo, this work establishes a robust means of screening libraries of LbL systems generated, affording simultaneous resolution over persistence and biodistribution of both the drug and carrier following systemic administration of a single particle formulation. Employing a PLGA drug-containing core as a substrate for LbL deposition, a range of coated systems were fabricated to investigate the abilities of these films to stabilize drug for delivery as well as to improve the pharmacokinetics of both the drug and carrier. Significant reductions in liver accumulation were observed for different formulations of the layered architectures within the first 30 min of systemic circulation. LbL architectures diminished liver localization of the surrogate drug, cardiogreen, by 10–25% ID/g relative to native PLGA nanoparticles and modulated carrier accumulation in the liver >50% ID/g. Further, enhanced persistence of the drug was observed with the coated systems, significantly increasing the drug half-life from 2 to 3 min for free drug and 1.87 h for the uncoated core to 4.17 h and 4.54 h for the coated systems. These systems provide an exciting, modular platform that improves the pharmacokinetic properties of the therapeutic, reduces bolus release of drug from nanoparticles, and enhances the safety and circulation half-life of the drug in vivo, proving them to be highly clinically-relevant and a promising approach for future development of molecularly-targeted and combination therapeutics.Janssen Pharmaceutical Ltd. (TRANSCEND Partnership)National Science Foundation (U.S.). Graduate Research Fellowshi

Mechanism of Action of Azacytidine in Myelodysplastic Syndromes (MDS)

Abstract Introduction: Myelodysplastic syndromes (MDS) have historically been classified as a set of heterogeneous hematopoietic stem cell (HSC) disorders, which are characterized clinically by abnormalities in the hematopoietic system. However, several recent landmark studies have now demonstrated that the pathogenesis of MDS is not confined to HSCs, and mesenchymal stromal cells (MSCs) in the bone marrow also play important contributing roles in sustaining the disorder. Treatment for MDS using hypomethylating agents such as azacytidine is effective, with patients showing recovery of blood counts and long-term restoration of normal hematopoiesis - an outcome that is plausibly brought about only by the reversal of abnormalities the bone marrow stem cell niches. In this work, we investigate the use of azacytidine in both HSCs and MSCs of MDS patients in order to better understand its therapeutic mechanism on stem cell niches, as well as to inform strategies for the development of future therapies for similar hematopoietic disorders. Methods: Cryopreserved BM MDS samples (n=20) were obtained from the Department of Hematology repository at Singapore General Hospital. Healthy MSCs were derived from bone marrow aspirates of healthy donors, obtained at Singapore General Hospital. Healthy CD34+ HSCs were purchased from Lonza. Osteogeneic and adipogeneic differentiation capabilities and proliferation capacities were performed on MSCs. Proliferation, cell cycling and apoptosis in HSCs were analysed. Gene expression profiling for MDS candidate genes was performed by quantitative PCR on both MSCs and HSCs. Co-culture experiments with healthy CD34+ cells on MDS MSCs were investigated. All assays were performed on both MSCs and HSCs, before and after azacytidine treatment. Results: MDS MSCs have significantly reduced proliferative capacities (p=0.02) and osteogeneic differentiation potentials (p=0.0006) compared to healthy MSCs. Gene expression profiling of MDS MSCs showed a 4.6-fold (n=17; p=0.0002) and 6.2-fold (n=15; p=0.0002) reduction in osteogeneic markers like Runx2 and Osterix respectively. Hematopoietic growth factors and chemokines such as IGF1, IL-8 and Angiopoietin-1 are 5.35-fold (n=17; p&lt;0.0001), 3.36-fold (n=20; p=0.02) and 1.45-fold (n=15; p=0.2) lower than healthy controls. After treatment with azacytidine, MDS MSCs demonstrated significant increased proliferative capacities (n=4; p&lt;0.0001) and differentiation potentials (n=3; p&lt;0.0001) in comparison to healthy MSCs. Significant increase in gene expression of Osterix (n=5; p&lt;0.0001) was seen in comparison to healthy controls. In MDS HSCs, expression of hematopoietic, cell cycling and apoptosis genes such as CXCR4, CCL3, Cyclin D1 and BCL2 are significantly different from healthy HSC - 13 fold (n=15; p=0.1005), 6.8 fold (n=15; p=0.014), 20 fold (n=19; p=0.2673) and 5.26 fold (n=19; p=0.0478) lower than healthy HSCs, respectively. Proliferation of MDS HSCs in culture was 3.3 fold higher than healthy HSCs but treatment with azacytidine of 1µM and 5µM reduced the growth advantage of MDS HSCs to 3 fold and 4.2 fold in comparison with similarly treated healthy controls. Co-culture experiments of healthy CD34+ cells on MDS MSCs, induced a gene expression profile in healthy HSCs similar to MDS HSC. After treatment of MDS MSCs with azacytidine, the gene expression of expanded healthy CD34+ cells was normal. Conclusion: MDS stromal cells are functionally abnormal and have the ability to instruct healthy HSCs to adopt genetic features that resemble MDS HSCs. Treatment with azacytidine restores normal function to MDS MSCs while conferring a growth disadvantage to MDS HSCs but not healthy HSCs. These observations help elucidate for the first time a possible mechanism of action by azacytidine on stromal cells in the treatment of MDS and further suggest that therapies which also target stromal elements in bone marrow niches may be necessary in achieving more favorable outcomes for hematopoieic disorders such as MDS. Disclosures Hwang: Janssen-Cilag, Singapore: Honoraria, Other: Travel Support; Celgene, Singapore: Honoraria, Other: Travel Support; Roche, Singapore: Honoraria, Other: Travel Support; Pfizer, Singapore: Honoraria, Other: Travel Support; Novartis, Singapore: Honoraria, Other: Travel Support; BMS, Singapore: Honoraria, Other: Travel Support; MSD, Singapore: Honoraria, Other: Travel Support; Sanofi, Singapore: Honoraria, Other: Travel Support. </jats:sec