nanoparticle oral delivery by bile acid transporter...
TRANSCRIPT
Nanoparticle Oral Delivery by Bile Acid Transporter Mediation
Materials and Technology for Drug and Nucleic Acid Delivery
You Han Bae, University of Utah
Corresponding Author: You Han Bae, University of Utah
Abstract
Nano-sized drug delivery systems, which have mostly been introduced into the body via parenteral routes, may present
meritorious properties by altering the biodistribution of an active pharmaceutical ingredient (API) and accompanying
toxicity profiles. However their full potential is remaining to be proven in human patients even after a handful of phase
2-3 clinical trials have been reported. This presentation introduces an alternative route for the administration of
nanomedicine to avoid invasive delivery. There are numerous biopharmaceuticals, such as polynucleotides and
proteins/peptides, as well as small molecules which are suffering from nil or poor oral bioavailability. Major factors
determining oral bioavailability of a drug of interest include solubility, permeability, metabolism, digestion, and stomach
pH. If nanoparticles were absorbed as a whole in the gastrointestinal tract, it would avoid most of issues relevant to
poor bioavailability.
We are exploring potential approaches for the oral absorption of intact nanoparticles. One example is to use bile acid
transporters. Bile acids are secreted to help digest oily nutrients and recycled from the liver to gall bladder, to small
intestine, and back to the liver with high efficiency, which is named as ‘enterohepatic circulation.’ The recycling is
accomplished by the cells in the distal ileal segment and the liver, which express a series of specialized transporters.
Recent discovery in literature revealed that the transporters, evolved as molecular binders and pumps to effectively
recycle bile acids, serves as receptors to actively uptake heparin modified with deoxycholic acid (a bile acid) and help
deliver to the portal vein.1 This study expands the approach to nanoparticles which carry APIs, ranging from small
molecules, to peptides/proteins, and to pDNA. This presentation will highlight oral delivery of nanomedicine in mice,
rats and monkey and emphasize its significance.
References
1. T.A. Al-Hilal, S.W. Chung, F. Alam, J. Park, K.E. Lee, H. Jeon, K. Kim, I.C. Kwon, I.-S. Kim, S.Y. Kim, Y. Byun, Sci. Rep.
2014, 4, 4163.
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SWCNT localization in biological cells
Materials and Technology for Drug and Nucleic Acid Delivery
Tetyana Ignatova, Lehigh University; Massooma Pirbhai, Susquehanna University; Swetha Chandrasekar, Lehigh University; Slava V Rotkin, Lehigh University; Sabrina Jedlicka, Lehigh University
Corresponding Author: Sabrina Jedlicka, Materials Science and Engineering Department, Lehigh University
Abstract
The unique physical properties and dimensionality of single wall carbon nanotubes (SWCNTs) make them attractive for
biomedical applications and diagnostics in the field of cell biology. Several groups have already shown that various cell
types can uptake SWCNTs, suggesting their potential as delivery vehicles for biologically active cargo. However, there are
a number of questions that remain regarding cytotoxicity and SWCNT- cell interactions on both the cellular and
molecular level. Here we use Raman Spectroscopy to investigate C17.2 neural stem cells after uptake of SWCNTs
wrapped with ssDNA over a wide variety of time periods, allowing for precise localization of SWCNTs inside the cells over
extended time periods. The localization data is being used to understand how, upon uptake of SWCNTs, the
cytoskeleton and other cellular structures of the adherent cells are perturbed.
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Non-phospholipid liposomes for co-delivery of small molecular drugs and siRNA
Materials and Technology for Drug and Nucleic Acid Delivery
Min Lee, University of California, Los Angeles; Zhongkai Cui, University of California, Los Angeles; Tara Aghaloo, University of California, Los Angeles
Corresponding Author: Min Lee, UCLA
Abstract
The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple
signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small
molecule phenamil combined with suppression of natural bone morphogenetic protein (BMP) antagonists such as
noggin is a promising therapeutic strategy that enhances BMP signaling and bone repair. The binary mixtures of single-
chain amphiphiles and sterols have been shown to form fluid lamellar phases (non-phospholipid liposomes;
sterosomes). Compared with conventional phospholipid liposome, a distinct feature of these sterosomes is their high
sterol content (50-70 mol%) that induces well-ordered lipid bilayer chains and significantly increased nanoparticle
stability. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery
system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes
hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrated the
ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed
siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a
mouse calvarial defect model. SA/Chol Sterosomes retained their initial size and zeta potential during incubation under
various stressors (temperature, pH, organic solvent), indicating their high stability. SA/Chol supported sustained release
of phenamil and demonstrated osteogenic efficacy at significantly reduced drug dosage compared with treatment using
free phenamil. Furthermore, SA/Chol and siRNA complexes significantly enhanced cellular uptake and gene knockdown
efficiency in MSCs with minimal cytotoxicity compared with commercially available lipofectamine 2000. Lastly, co-
delivery of phenamil + noggin siRNA in the Sterosomes synergistically enhanced MSC osteogenesis in hydrogels and
calvarial bone formation in vivo compared with Sterosomes loaded with phenamil or siRNA alone. Since current
liposomal biomaterials are not truly inductive and have no intrinsic therapeutic effects, we are currently developing a
new Sterosome formulation with osteoinductive properties by modulating the selection of sterol, one of the Sterosome
components. This study suggests a new non-phospholipid liposomal platform with osteoinductive properties for delivery
of small molecular drugs and/or therapeutic genes for enhanced bone formation.
Cationic Polymeric Micelle as a drug and siRNA Carrier for Axonal Regeneration after rat compression SCI
Materials and Technology for Drug and Nucleic Acid Delivery
So Jung Gwak, Clemson University; Christia Macks, Clemson University; Mark Kindy, University of South Florida; Michael Lynn, Greenville Health System; Ken Webb, Clemson University; and Jeoung Soo Lee, Clemson University
Corresponding Author: Jeoung Soo Lee, Clemson University
Abstract
Introduction: The regenerative capacity of the injured adult CNS is extremely limited due to both extrinsic
microenvironmental factors and intrinsic, age-related changes in neuronal biochemistry. Many studies have shown that
diverse extracellular inhibitors of neuroplasticity including both myelin associated inhibitors (MAIs) and chondroitin
sulfate proteoglycans (CSPGs) may act through common intracellular signaling pathways. Neurite growth inhibition in
response to MAIs and CSPGs has been shown to be associated with activation of RhoA and Rho kinase (ROCK) and can
be overcome by Rho/ROCK inhibitors. Our goal is to develop cationic polymeric micelle nanoparticle for combinatorial
delivery of multiple bioactive molecules targeting different barriers to plasticity and axonal regeneration. We
synthesized novel cationic, amphiphilic copolymers (poly (lactide-co-glycolide)-g-polyethylenimine: PgP) that provides
efficient drug and nucleic acid delivery. Here, we show that PgP can deliver RhoA siRNA and efficiently knockdown RhoA
gene expression in a rat compression spinal cord injury model in vivo. We also show that PgP can deliver Rolipram (Rm)
can reduce inflammaroty response and apoptosis after compression SCI.
Methods: To generate rat compression SCI model, laminectomy was performed on the back of Sprague Dawley rats and
the T9 spinal cord region was exposed. PgP/RhoA siRNA polyplexes (20 µg RhoA siRNA) were prepared and injected at
the T9 spinal cord injury region. At 1, 2, and 4 weeks after polyplex injection, the rats were sacrificed and total RNA was
isolated and RhoA gene knockdown is measured by real-time PCR. For histological evaluation, rat were perfused with 4%
paraformaldehyde at 4 weeks after injection of PgP/RhoA siRNA and then spinal cords were retrieved and sectioned
longitudinally. The sections were stained using antibodies against neurofilament and GFAP and digitally imaged using an
inverted Epifluorescent microscope. Rolipram was loaded in hydrophobic core of PgP by solvent evaporation method
and the loading efficiency was evaluated by HPLC. Rm-loaded PgP (10 µl, 10 µg Rm) was intraspinally injected in the
injured spinal cord. For histological evaluation, rat were cardiac perfused and then spinal cords were retrieved and
sectioned longitudinally. The sections were analyzed by TUNEL assay and stained using antibodies against ED1 and
digitally imaged using an inverted Epifluorescent microscope.
Results: In untreated SCI animal group, RhoA gene expression was increased 2.61-fold compared to that in sham animal
group at 7 days. In PgP/RhoA siRNA nanoparticle treated SCI animal groups, the RhoA expression was reduced to levels
not significantly different from the sham control group and significant knockdown was maintained up to 4 weeks. We
also observed an extensive necrotic lesion cavity and significant reactive astrogliosis in the spinal cord from untreated
SCI animal group, while reduced cavitation/astrogliosis and axonal regeneration into the lesion site in spinal cord from
PgP/RhoA siRNA polyplex-treated animal group was observed. Rm delivery by PgP (Rm-PgP) restored cAMP level to that
in sham animal group and reduced the inflammatory response and apotosis.
Discussion and Conclusion: These studies demonstrate that PgP is a promising co-delivery carrier for rolipram and
therapeutic siRNA in rat compression spinal cord injury model in vivo. Currently, we are evaluating the effect of co
delivery of Rm-PgP and PgP/RhoA siRNA on functional recovery by Basso-Beattie-Bresnahan (BBB) locomotor rating
scale and contact placing response in rat compression SCI model.
Acknowledgements: Research reported in this publication was supported by NIGMS of the NIH under award number
5P20GM103444-07 and South Carolina Spinal Cord Injury Fund under award number SCIRF # 2014 I-02.
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Constructing Homogeneous Spherical Nucleic Acid Aptamer Nanoparticles as Drug Delivery Vehicles for Treating Cancer
Materials and Technology for Drug and Nucleic Acid Delivery
Ricky Whitener, Auburn University; Madison Nash, Auburn University; Jacek Wower, Auburn University; Mark Byrne, Rowan University
Corresponding Author: Ricky Whitener, Auburn University
Abstract
Rationale
Nucleic acid aptamers, due to recent advances in SELEX technology, have been developed on a rapid basis the past few
years. However, only one aptamer has successfully made it to the market, and a few are finishing clinical trials, but most
of these aptamers are not being considered for use as a therapeutic tool. This is because a single aptamer sequence
does not have just one homogeneous structure. Our approach is to use a combination of engineering knowledge on
hybridization stability and modeling, along with biochemistry expertise, to develop aptamers that specifically bind to our
developed nanocarrier platform. It is our hope that stable aptamers will have an improved chance to be successful in
clinical trials.
Objectives
The main goal of our research is to analyze the assembly of aptamers and drugs onto AuNps resulting in the creation of a
highly controlled and regulated protocol for producing a spherical DNA nanoparticle construct. The different proponents
of the goal will result in making a generalized DNA platform that is applicable for a variety of newly developed aptamers.
Also, it will allow for an optimal amount of both aptamers and drug attachment to the AuNps.
Methods
Our nanocarrier platform consists of a DNA strand, called an “anchor”, that is able to hybridize with an aptamer strand
and covalently bind to a 15nm AuNp due to a thiol modification on its 5’ end. The area where hybridization occurs is the
drug-binding module. A 15nm AuNp is chosen because the total size of our nanocarrier platform will be in the range for
passive targeting, in addition to active targeting induced by the aptamer. AuNps are also known to be non-toxic and
inert in vivo. They are also suitable for imaging as they are detectable by x-rays.
Results
Our data demonstrate that we are able to specifically regulate the number of anchor strands bound to the AuNp by
manipulating the DNA and salt concentrations during the nanoparticle functionalization process. Furthermore, we are
able to modulate the release of the chemotherapeutic drug daunomycin by “mutating” nucleic acid strand sequences
that intercalate the drug.
In vitro results indicate cytotoxicity of our drug-loaded nanocarrier is comparable to that of free drug. This was tested by
assessing cell viability of MCF-7 breast cancer cells treated with 0-1000nM nanocarrier and free drug with an XTT assay
and live/dead staining. Our nanocarrier is shown to efficiently release drug over an extended time period, while
obtaining ~98% cell death. Furthermore, our engineered aptamer is capable of specifically targeting NCI-H69 small cell
lung cancer cells (SCLC) over other cell lines.
Conclusions
Creating an AuNp DNA construct with a specific structure in mind is difficult due to the high sensitivity of aptamers in
differing buffers, salt concentrations, temperatures, etc… By understanding these molecules we have optimized specific
conditions to create a homogeneous nanocarrier. Our engineered aptamer is capable of specifically recognizing SCLC
cells compared to other cell lines and maintains its functionality when attached to our nanocarrier.
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Controlled Delivery of a Therapeutic Glycosaminoglycan for Amelioration of Radiation-Induced Proctitis-Associated Pain
Materials and Technology for Drug and Nucleic Acid Delivery
Mark Martin Jensena,b, Wanjian Jiac,, Kyle J. Isaacsona,b, Austin Schultsc, Joseph Cappellod, Glenn D. Prestwiche, Siam Oottamasathienc,f*, Hamidreza Ghandeharia,b,d*
aDepartment of Bioengineering, University of Utah, Salt Lake City, UT, 84112 USA bUtah Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, UT, 84112 USA cDivision of Urology, Section of Pediatric Urology, University of Utah, Salt Lake City, UT, 84113 USA dDepartment of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, 84112 USA eDepartment of Medicinal Chemistry, University of Utah, Salt Lake City, UT, 84112 USA fDepartment of Surgery and Division of Pediatric Urology, Primary Children’s Hospital, Salt Lake City, UT, 84113 USA
Pain and discomfort from Radiation-induced proctitis (RIP) are among the most common clinical adverse events for patients receiving radiotherapy as part of treatment for ovarian, prostate, colon, and bladder cancer. RIP limits radiation dosage, interrupts treatment, and lowers patients’ quality of life. Current treatments for RIP are reactionary and typically administered only after the onset of RIP and are only sparingly successful in ameliorating RIP symptomatology. No effective prophylactic treatment options exist for RIP in spite of its prevalence and clinical significance. A prophylactic treatment that protects the gastrointestinal tract from the deleterious effects of radiotherapy will improve patient quality of life and may allow for higher doses of radiation to be administered more regularly, leading to improved clinical outcome. We report the development of a novel prophylactic treatment using two bioinspired polymers, i.e., an anti-inflammatory semi-synthetic glycosaminoglycan (GAG) GM-0111, generated from the sulfation of hyaluronic acid, and a recombinant protein polymer silk-elastinlike protein polymer (SELPs) 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit). The combination of these materials created a thermoresponsive in situ gelling rectal delivery system with an injectable viscosity, a <5 minute gelation time, a final modulus that permits peristaltic elimination, and a release profile that enhances accumulation of GM-0111 within the lining of the rectum. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21 days by 53 ± 4%, 47 ± 10%, and 12 ± 6%, respectively . Also, SELP 815K alone significantly reduced pain by 24 ± 10% after 3 days and 19 ± 10% after 7 days. SELP in situ gelling polymers enhance the therapeutic efficacy of GAG GM-0111, reducing post-irradiation pathology and pain, which could significantly improve the effectiveness of radiation treatment and the quality of life for cancer patients.