nanoparticle oral delivery by bile acid transporter...

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.

D

SWCNT localization in biological cells


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.

D

Non-phospholipid liposomes for co-delivery of small molecular drugs and siRNA


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


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.

D

Constructing Homogeneous Spherical Nucleic Acid Aptamer Nanoparticles as Drug Delivery Vehicles for Treating Cancer


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.

D

Controlled Delivery of a Therapeutic Glycosaminoglycan for Amelioration of Radiation-Induced Proctitis-Associated Pain


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.

nanoparticle oral delivery by bile acid transporter...

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