Publications by authors named "Shashank R Sirsi"

28 Publications

  • Page 1 of 1

Non-invasive molecularly-specific millimeter-resolution manipulation of brain circuits by ultrasound-mediated aggregation and uncaging of drug carriers.

Nat Commun 2020 10 1;11(1):4929. Epub 2020 Oct 1.

Institute of Neuroinformatics, D-ITET, ETH Zurich and UZH, Zurich, Switzerland.

Non-invasive, molecularly-specific, focal modulation of brain circuits with low off-target effects can lead to breakthroughs in treatments of brain disorders. We systemically inject engineered ultrasound-controllable drug carriers and subsequently apply a novel two-component Aggregation and Uncaging Focused Ultrasound Sequence (AU-FUS) at the desired targets inside the brain. The first sequence aggregates drug carriers with millimeter-precision by orders of magnitude. The second sequence uncages the carrier's cargo locally to achieve high target specificity without compromising the blood-brain barrier (BBB). Upon release from the carriers, drugs locally cross the intact BBB. We show circuit-specific manipulation of sensory signaling in motor cortex in rats by locally concentrating and releasing a GABA receptor agonist from ultrasound-controlled carriers. Our approach uses orders of magnitude (1300x) less drug than is otherwise required by systemic injection and requires very low ultrasound pressures (20-fold below FDA safety limits for diagnostic imaging). We show that the BBB remains intact using passive cavitation detection (PCD), MRI-contrast agents and, importantly, also by sensitive fluorescent dye extravasation and immunohistochemistry.
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http://dx.doi.org/10.1038/s41467-020-18059-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529901PMC
October 2020

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake .

Theranostics 2020 9;10(18):8143-8161. Epub 2020 Jul 9.

Department of Biomedical Engineering, University of Texas at Dallas, Richardson, TX, USA.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with "microbubble" ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. : To monitor the therapeutic efficacy of sonopermeation we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted along with associated vascular changes. : In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment . Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. : Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by qCEUS imaging and analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study.
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http://dx.doi.org/10.7150/thno.45903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381728PMC
July 2020

Formulation and Characterization of Chemically Cross-linked Microbubble Clusters.

Langmuir 2019 08 7;35(33):10977-10986. Epub 2019 Aug 7.

University of Texas at Dallas , Richardson , Texas , 75080 , United States.

The purpose of this study is to introduce a new concept of chemically cross-linked microbubble clusters (CCMCs), which are individual microbubble ultrasound contrast agents (UCAs) physically tethered together. We demonstrate a facile means of their production, characterize their size and stability, and describe how they can potentially be used in biomedical applications. By tethering UCAs together into CCMCs, we propose that novel methods of ultrasound mediated imaging and therapy can be developed through unique interbubble interactions in an ultrasound field. One of the major challenges in generating CCMCs is controlling aggregate sizes and maintaining stability against Ostwald ripening and coalescence. In this study, we demonstrate that chemically cross-linked microbubble clusters can produce small (<10 μm) quasi-stable complexes that slowly fuse into bubbles with individual gas cores. Furthermore, we demonstrate that this process can be driven with low-intensity ultrasound pulses, enabling a rapid fusion of clusters which could potentially be used to develop novel ultrasound contrast imaging and drug delivery strategies in future studies. The development of novel microbubble clusters presents a simple yet robust process for generating novel UCAs with a design that could allow for more versatility in contrast-enhanced ultrasound (CEUS), molecular imaging, and drug delivery applications. Additionally, microbubble clustering is a unique way to control size, shell, and gas compositions that can be used to study bubble ripening and coalescence in a highly controlled environment or study the behavior of mixed-microbubble populations.
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http://dx.doi.org/10.1021/acs.langmuir.9b00475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061884PMC
August 2019

Impact of hydrostatic pressure on phase-change contrast agent activation by pulsed ultrasound.

J Acoust Soc Am 2019 06;145(6):3457

Department of Bioengineering, University of Texas at Dallas, Richardson, Texas 75080, USA.

A phase-change contrast agent (PCCA) can be activated from a liquid (nanodroplet) state using pulsed ultrasound (US) energy to form a larger highly echogenic microbubble (MB). PCCA activation is dependent on the ambient pressure of the surrounding media, so any increase in hydrostatic pressure demands higher US energies to phase transition. In this paper, the authors explore this basic relationship as a potential direction for noninvasive pressure measurement and foundation of a unique technology the authors are developing termed tumor interstitial pressure estimation using ultrasound (TIPE-US). TIPE-US was developed using a programmable US research scanner. A custom scan sequence interleaved pulsed US transmissions for both PCCA activation and detection. An automated US pressure sweep was applied, and US images were acquired at each increment. Various hydrostatic pressures were applied to PCCA samples. Pressurized samples were imaged using the TIPE-US system. The activation threshold required to convert PCCA from the liquid to gaseous state was recorded for various US and PCCA conditions. Given the relationship between the hydrostatic pressure applied to the PCCA and US energy needed for activation, phase transition can be used as a surrogate of hydrostatic pressure. Consistent with theoretical predictions, the PCCA activation threshold was lowered with increasing sample temperature and by decreasing the frequency of US exposure, but it was not impacted by PCCA concentration.
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http://dx.doi.org/10.1121/1.5111345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570615PMC
June 2019

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

J Clin Invest 2018 01 27;128(1):309-322. Epub 2017 Nov 27.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial FcγRIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via FcγRIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of FcγRIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial FcγRIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.
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http://dx.doi.org/10.1172/JCI89333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749535PMC
January 2018

Ultrasound-Stimulated Drug Delivery Using Therapeutic Reconstituted High-Density Lipoprotein Nanoparticles.

Nanotheranostics 2017 1;1(4):440-449. Epub 2017 Nov 1.

Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080 USA.

The abnormal tumor vasculature and the resulting abnormal microenvironment are major barriers to optimal chemotherapeutic drug delivery. It is well known that ultrasound (US) can increase the permeability of the tumor vessel walls and enhance the accumulation of anticancer agents. Reconstituted high-density lipoproteins (rHDL) nanoparticles (NPs) allow selective delivery of anticancer agents to tumor cells via their overexpressed scavenger receptor type B1 (SR-B1) receptor. The goal of this study is to investigate the potential of noninvasive US therapy to further improve delivery and tumor uptake of the payload from rHDL NPs, preloaded with an infrared dye (IR-780), aimed to establish a surrogate chemotherapeutic model with optical localization. Athymic nude mice were implanted orthotopically with one million breast cancer cells (MDA-MB-231/Luc). Three weeks later, animals were divided into seven groups with comparable mean tumor size: control, low, moderate, and high concentration of rHDL NPs alone groups, as well as these three levels of rHDL NPs plus US therapy groups ( = 7 to 12 animals per group), where low, moderate and high denote 5, 10, and 50 µg of the IR-780 dye payload per rHDL NP injection, respectively. The US therapy system included a single element focused transducer connected in series with a function generator and power amplifier. A custom 3D printed cone with an acoustically transparent aperture and filled with degassed water allowed delivery of focused US energy to the tumor tissue. US exposure involved a pulsed sequence applied for a duration of 5 min. Each animal in the US therapy groups received a slow bolus co-injection of MB contrast agent and rHDL NPs. Animals were imaged using a whole-body optical system to quantify intratumoral rHDL NP accumulation at baseline and again at 1 min, 30 min, 24 h, and 48 h. At 48 h, all animals were euthanized and tumors were excised for analysis. We investigated a noninvasive optical imaging method for monitoring the effects of US-stimulated drug delivery of IR-780 dye-loaded rHDL NPs in living animals. No change in optical imaging data was found in the control animals. However, there was considerable dye accumulation (surrogate drug) within 48 h in the low (5 µg), moderate (10 µg), and high (50 µg) rHDL NP concentration-dosed group animals ( < 0.09). With US therapy added to the experimental protocol, there was an additional and significant increase in local tumor drug uptake at 48 h ( < 0.02). Optical image data collected from tumor samples confirmed tumor retention of the IR-780 dye-loaded rHDL NPs and correlated positively with optical imaging results ( > 0.69, < 0.003). IR-780 dye extraction from the tumor tissue samples confirmed the and US therapy findings. Overall, the addition of US therapy considerably improved local rHDL NP accumulation in tumor tissue. This study concludes that US-mediated drug delivery can facilitate tumor uptake of rHDL NPs and more research is warranted to optimize the drug dosing schedule and the respective therapeutic protocols.
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http://dx.doi.org/10.7150/ntno.21905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704009PMC
November 2017

Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis.

ACS Appl Mater Interfaces 2017 Nov 20;9(43):37587-37596. Epub 2017 Oct 20.

Department of Radiology, Translational Research in Ultrasound Theranostics (TRUST) Program, University of Texas Southwestern Medical Center , 5323 Harry Hines Boulevard, Dallas, Texas 75390-8514, United States.

Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.
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http://dx.doi.org/10.1021/acsami.7b10592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691601PMC
November 2017

Toward optimization of in vivo super-resolution ultrasound imaging using size-selected microbubble contrast agents.

Med Phys 2017 Dec 27;44(12):6304-6313. Epub 2017 Oct 27.

Department of Bioengineering, University of Texas at Dallas, Richardson, TX, 75080, USA.

Purpose: Microvascular processes play key roles in many diseases including diabetes. Improved understanding of the microvascular changes involved in disease development could offer crucial insight into the relationship of these changes to disease pathogenesis. Super-resolution ultrasound (SR-US) imaging has showed the potential to visualize microvascular detail down to the capillary level (i.e., subwavelength resolution), but optimization is still necessary. The purpose of this study was to investigate in vivo SR-US imaging of skeletal muscle microvascularity using microbubble (MB) contrast agents of various size and concentration while evaluating different ultrasound (US) system level parameters such as imaging frame rate and image acquisition length.

Methods: An US system equipped with a linear array transducer was used in a harmonic imaging mode at low transmit power. C57BL/6J mice fed a normal diet were used in this study. An assortment of size-selected MB contrast agents (1-2 μm, 3-4 μm, and 5-8 μm in diameter) were slowly infused in the tail vein at various doses (1.25 × 10 , 2.5 × 10 , or 5 × 10  MBs). US image data were collected before MB injection and thereafter for 10 min at 30 frames per s (fps). The US transducer was fixed throughout and between each imaging period to help capture microvascular patterns along the same image plane. An adaptive SR-US image processing technique was implemented using custom Matlab software.

Results: Experimental findings illustrate the use of larger MB results in better SR-US images in terms of skeletal muscle microvascular detail. A dose of 2.5 × 10  MBs resulted in SR-US images with optimal spatial resolution. An US imaging rate of at least 20 fps and image acquisition length of at least 8 min also resulted in SR-US images with pronounced microvascular detail.

Conclusions: This study indicates that MB size and dose and US system imaging rate and data acquisition length have significant impact on the quality of in vivo SR-US images of skeletal muscle microvascularity.
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http://dx.doi.org/10.1002/mp.12606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734623PMC
December 2017

Condensation phase diagrams for lipid-coated perfluorobutane microbubbles.

Langmuir 2014 Jun 21;30(21):6209-18. Epub 2014 May 21.

Department of Mechanical Engineering, University of Colorado , Boulder, Colorado 80309, United States.

The goal of this study was to explore the thermodynamic conditions necessary to condense aqueous suspensions of lipid-coated gas-filled microbubbles into metastable liquid-filled nanodrops as well as the physicochemical mechanisms involved with this process. Individual perfluorobutane microbubbles and their lipid shells were observed as they were pressurized at 34.5 kPa s(-1) in a microscopic viewing chamber maintained at temperatures ranging from 5 to 75 °C. The microbubbles contracted under pressure, ultimately leading to either full dissolution or microbubble-to-nanodrop condensation. Temperature-pressure phase diagrams conveying condensation and stability transitions were constructed for microbubbles coated with saturated diacylphosphatidylcholine lipids of varying acyl chain length (C16 to C24). The onset of full dissolution was shifted to higher temperatures with the use of longer acyl chain lipids or supersaturated media. Longer chain lipid shells resisted both dissolution of the gas core and mechanical compression through a pronounced wrinkle-to-fold collapse transition. Interestingly, the lipid shell also provided a mechanical resistance to condensation, shifting the vapor-to-liquid transition to higher pressures than for bulk perfluorobutane. This result indicated that the lipid shell can provide a negative apparent surface tension under compression. Overall, the results of this study will aid in the design and formulation of vaporizable fluorocarbon nanodrops for various applications, such as diagnostic ultrasound imaging, targeted drug delivery, and thermal ablation.
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http://dx.doi.org/10.1021/la501004uDOI Listing
June 2014

State-of-the-art materials for ultrasound-triggered drug delivery.

Adv Drug Deliv Rev 2014 Jun 31;72:3-14. Epub 2013 Dec 31.

Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309, USA; Materials Science and Engineering Program, University of Colorado, Boulder, CO 80309, USA. Electronic address:

Ultrasound is a unique and exciting theranostic modality that can be used to track drug carriers, trigger drug release and improve drug deposition with high spatial precision. In this review, we briefly describe the mechanisms of interaction between drug carriers and ultrasound waves, including cavitation, streaming and hyperthermia, and how those interactions can promote drug release and tissue uptake. We then discuss the rational design of some state-of-the-art materials for ultrasound-triggered drug delivery and review recent progress for each drug carrier, focusing on the delivery of chemotherapeutic agents such as doxorubicin. These materials include nanocarrier formulations, such as liposomes and micelles, designed specifically for ultrasound-triggered drug release, as well as microbubbles, microbubble-nanocarrier hybrids, microbubble-seeded hydrogels and phase-change agents.
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http://dx.doi.org/10.1016/j.addr.2013.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041842PMC
June 2014

In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.

Mol Imaging 2013 Sep;12(6):357-63

Department of Mechanical Engineering, University of Colorado, CO 8030, USA.

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494687PMC
September 2013

Lung surfactant microbubbles increase lipophilic drug payload for ultrasound-targeted delivery.

Theranostics 2013 20;3(6):409-19. Epub 2013 May 20.

Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309, USA.

The cavitation response of circulating microbubbles to targeted ultrasound can be used for noninvasive, site-specific delivery of shell-loaded materials. One challenge for microbubble-mediated delivery of lipophilic compounds is the limitation of drug loading into the microbubble shell, which is commonly a single phospholipid monolayer. In this study, we investigated the use of natural lung surfactant extract (Survanta(®), Abbott Nutrition) as a microbubble shell material in order to improve drug payload and delivery. Pulmonary surfactant extracts such as Survanta contain hydrophobic surfactant proteins (SP-B and SP-C) that facilitate lipid folding and retention on lipid monolayers. Here, we show that Survanta-based microbubbles exhibit wrinkles in bright-field microscopy and increased lipid retention on the microbubble surface in the form of surface-associated aggregates observed with fluorescence microscopy. The payload of a model lipophilic drug (DiO), measured by flow cytometry, increased by over 2-fold compared to lipid-coated microbubbles lacking SP-B and SP-C. Lung surfactant microbubbles were highly echogenic to contrast enhanced ultrasound imaging at low acoustic intensities. At higher ultrasound intensity, excess lipid was observed to be acoustically cleaved for localized release. To demonstrate targeting, a biotinylated lipopolymer was incorporated into the shell, and the microbubbles were subjected to a sequence of radiation force and fragmentation pulses as they passed through an avidinated hollow fiber. Lung surfactant microbubbles showed a 3-fold increase in targeted deposition of the model fluorescent drug compared to lipid-only microbubbles. Our results demonstrate that lung surfactant microbubbles maintain the acoustic responsiveness of lipid-coated microbubbles with the added benefit of increased lipophilic drug payload.
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http://dx.doi.org/10.7150/thno.5616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677411PMC
January 2014

Advances in ultrasound mediated gene therapy using microbubble contrast agents.

Theranostics 2012 31;2(12):1208-22. Epub 2012 Dec 31.

Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309, USA.

Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.
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http://dx.doi.org/10.7150/thno.4306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563148PMC
October 2013

Microbubbles as biocompatible porogens for hydrogel scaffolds.

Acta Biomater 2012 Dec 3;8(12):4334-41. Epub 2012 Aug 3.

Cooper Union, Department of Mechanical Engineering, 41 Cooper Square, New York, NY 10003, USA.

In this study, we explored the application of lipid-shelled, gas-filled microbubbles as a method for creating on-demand microporous hydrogels for cartilage tissue engineering. The technique allowed for homogenous distribution of cells and micropores within the scaffold, increasing the absorption coefficient of large solutes (70kDa dextran) over controls in a concentration-dependent manner. The stability of the gas phase of the microbubbles depended on several factors, including the initial size distribution of the microbubble suspension, as well as the temperature and pressure during culture. Application of pressure cycles provided controlled release of the gas phase to generate fluid-filled micropores with remnant lipid. The resulting microporous agarose scaffolds were biocompatible, leading to a twofold increase in engineered cartilage properties (E(Y)=492±42kPa for the bubble group vs. 249±49kPa for the bubble-free control group) over a 42-day culture period. Our results suggest that microbubbles offer a simple and robust method of modulating mass transfer in cell-seeded hydrogels through mild pressurization, and the methodology may be expanded in the future to include focused ultrasound for improved spatio-temporal control.
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http://dx.doi.org/10.1016/j.actbio.2012.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654399PMC
December 2012

Contrast ultrasound imaging for identification of early responder tumor models to anti-angiogenic therapy.

Ultrasound Med Biol 2012 Jun 16;38(6):1019-29. Epub 2012 Mar 16.

Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309, USA.

Agents targeting vascular endothelial growth factor (VEGF) have been validated as cancer therapeutics, yet efficacy can differ widely between tumor types and individual patients. In addition, such agents are costly and can have significant toxicities. Rapid noninvasive determination of response could provide significant benefits. We tested if response to the anti-VEGF antibody bevacizumab (BV) could be detected using contrast-enhanced ultrasound imaging (CEUS). We used two xenograft model systems with previously well-characterized responses to VEGF inhibition, a responder (SK-NEP-1) and a non-responder (NGP), and examined perfusion-related parameters. CEUS demonstrated that BV treatment arrested the increase in blood volume in the SK-NEP-1 tumor group only. Molecular imaging of α(V)β(3) with targeted microbubbles was a more sensitive prognostic indicator of BV efficacy. CEUS using RGD-labeled microbubbles showed a robust decrease in α(V)β(3) vasculature following BV treatment in SK-NEP-1 tumors. Paralleling these findings, lectin perfusion assays detected a disproportionate pruning of smaller, branch vessels. Therefore, we conclude that the response to BV can be identified soon after initiation of treatment, often within 3 days, by use of CEUS molecular imaging techniques. The use of a noninvasive ultrasound approach may allow for earlier and more effective determination of efficacy of antiangiogenic therapy.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2012.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348332PMC
June 2012

Monitoring early tumor response to drug therapy with diffuse optical tomography.

J Biomed Opt 2012 Jan;17(1):016014

Columbia University, New York, Department of Biomedical Engineering, New York, New York 10027, USA.

Although anti-angiogenic agents have shown promise as cancer therapeutics, their efficacy varies between tumor types and individual patients. Providing patient-specific metrics through rapid noninvasive imaging can help tailor drug treatment by optimizing dosages, timing of drug cycles, and duration of therapy-thereby reducing toxicity and cost and improving patient outcome. Diffuse optical tomography (DOT) is a noninvasive three-dimensional imaging modality that has been shown to capture physiologic changes in tumors through visualization of oxygenated, deoxygenated, and total hemoglobin concentrations, using non-ionizing radiation with near-infrared light. We employed a small animal model to ascertain if tumor response to bevacizumab (BV), an anti-angiogenic agent that targets vascular endothelial growth factor (VEGF), could be detected at early time points using DOT. We detected a significant decrease in total hemoglobin levels as soon as one day after BV treatment in responder xenograft tumors (SK-NEP-1), but not in SK-NEP-1 control tumors or in non-responder control or BV-treated NGP tumors. These results are confirmed by magnetic resonance imaging T2 relaxometry and lectin perfusion studies. Noninvasive DOT imaging may allow for earlier and more effective control of anti-angiogenic therapy.
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http://dx.doi.org/10.1117/1.JBO.17.1.016014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380816PMC
January 2012

Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles.

Ultrasound Med Biol 2012 Mar;38(3):492-503

Department of Chemical Engineering, Columbia University, New York, NY, USA.

Ultrasound molecular imaging is a powerful diagnostic modality using microbubbles coated with targeting ligands specific for endothelial biomarkers. The circulation persistence of ligand-bearing contrast agents is a key determinant in their contrast enhancement and targeting capability. Prior studies have shown that targeted microbubbles with ligands attached to the shell using the conventional exposed-ligand architecture (ELA) could trigger undesired ligand-induced complement activation and decreased circulation time. Microbubbles with the buried-ligand architecture (BLA), however, were found to inhibit complement activation and prolong circulation time. In the present study, we extended the stealth BLA microbubble design to size-selected (4 to 5-μm diameter) microbubbles targeted with cyclic RGD peptide using the postlabeling technique. Microbubble circulation persistence was measured in the healthy mouse kidney using a Visualsonics Vevo 770 scanner operating at 40 MHz in fundamental mode. The circulation persistence for targeted BLA microbubbles was significantly longer compared with their ELA counterparts and similar to no-ligand controls. Use of the BLA instead of the ELA increased the circulation half-life approximately two-fold. Analysis of the time-intensity and time-fluctuation curves with a two-compartment pharmacokinetic model showed a minimal degree of nonspecific vascular adhesion for any group. These results demonstrate the importance of surface architecture in the design of targeted microbubbles for ultrasound molecular imaging.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2011.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273728PMC
March 2012

Theranostic Gd(III)-lipid microbubbles for MRI-guided focused ultrasound surgery.

Biomaterials 2012 Jan 10;33(1):247-55. Epub 2011 Oct 10.

Department of Chemical Engineering, Columbia University, NY 10027, USA.

We have synthesized a biomaterial consisting of Gd(III) ions chelated to lipid-coated, size-selected microbubbles for utility in both magnetic resonance and ultrasound imaging. The macrocyclic ligand DOTA-NHS was bound to PE headgroups on the lipid shell of pre-synthesized microbubbles. Gd(III) was then chelated to DOTA on the microbubble shell. The reaction temperature was optimized to increase the rate of Gd(III) chelation while maintaining microbubble stability. ICP-OES analysis of the microbubbles determined a surface density of 7.5 × 10(5) ± 3.0 × 10(5) Gd(III)/μm(2) after chelation at 50 °C. The Gd(III)-bound microbubbles were found to be echogenic in vivo during high-frequency ultrasound imaging of the mouse kidney. The Gd(III)-bound microbubbles also were characterized by magnetic resonance imaging (MRI) at 9.4 T by a spin-echo technique and, surprisingly, both the longitudinal and transverse proton relaxation rates were found to be roughly equal to that of no-Gd(III) control microbubbles and saline. However, the relaxation rates increased significantly, and in a dose-dependent manner, after sonication was used to fragment the Gd(III)-bound microbubbles into non-gas-containing lipid bilayer remnants. The longitudinal (r(1)) and transverse (r(2)) molar relaxivities were 4.0 ± 0.4 and 120 ± 18 mM(-1)s(-1), respectively, based on Gd(III) content. The Gd(III)-bound microbubbles may find application in the measurement of cavitation events during MRI-guided focused ultrasound therapy and to track the biodistribution of shell remnants.
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http://dx.doi.org/10.1016/j.biomaterials.2011.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030396PMC
January 2012

Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors.

J Control Release 2012 Jan 17;157(2):224-34. Epub 2011 Sep 17.

Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309, USA.

Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection.
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http://dx.doi.org/10.1016/j.jconrel.2011.09.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822338PMC
January 2012

Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides.

J Nanobiotechnology 2009 Apr 7;7. Epub 2009 Apr 7.

Drexel University College of Medicine, Department of Pharmacology and Physiology, Philadelphia, Pennsylvania 19102, USA.

Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD.
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http://dx.doi.org/10.1186/1477-3155-7-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671478PMC
April 2009

Polymersome delivery of siRNA and antisense oligonucleotides.

J Control Release 2009 Mar 12;134(2):132-40. Epub 2008 Nov 12.

Department of Chemical and Biomolecular Engineering and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, USA.

siRNA and antisense oligonucleotides, AON, have similar size and negative charge and are often packaged for in vitro delivery with cationic lipids or polymers-but exposed positive charge is problematic in vivo. Here we demonstrate loading and functional delivery of RNAi and AON with non-ionic, nano-transforming polymersomes. These degradable carriers are taken up passively by cultured cells after which the vesicles transform into micelles that allow endolysosomal escape and delivery of either siRNA into cytosol for mRNA knockdown or else AON into the nucleus for exon skipping within pre-mRNA. Polymersome-mediated knockdown appears as efficient as common cationic-lipid transfection and about half as effective as Lenti-virus after sustained selection. For AON, initial results also show that intramuscular injection into a mouse model of muscular dystrophy leads to the expected protein expression, which occurs along the entire length of muscle. The lack of cationic groups in antisense polymersomes together with initial tests of efficacy suggests broader utility of these non-viral carriers.
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http://dx.doi.org/10.1016/j.jconrel.2008.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740336PMC
March 2009

PEG-PEI copolymers for oligonucleotide delivery to cells and tissues.

Methods Mol Biol 2008 ;433:141-58

Basic and Clinical Myology Laboratory Department of Physiology, The University of Melbourne, Victoria, Australia.

Inefficient delivery of antisense oligonucleotides (AO) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) are extremely well-studied compounds that markedly improve the in vitro and in vivo delivery of AOs to cells and tissues. By varying the Mw of PEI, as well as the nature of PEG shielding, PEG-PEI-AO nanoparticulates can be prepared with a dynamic range of size, surface charge, and stability. Each of these properties in-turn influences the transfection capacity of the PEG-PEI-AO polyplexes. In addition, PEG-PEI copolymers are readily functionalized for enhanced efficacy and specificity of cellular and tissue targeting. The synthesis and functionalization of PEG-PEI copolymers is remarkably simple and requires very little specialized equipment. Thus, PEG-PEI copolymers represent a tractable and adaptable oligonucleotide delivery system that can be customized and optimized to the investigators' specific application. This chapter describes the step-by-step synthesis of several PEG-PEI copolymers that are specifically formulated to provide effective delivery of AOs using both in vitro and in vivo applications. We describe the preparation of the PEG-PEI-AO polyplexes and provide examples showing transfection of cultured cells in vitro, as well as skeletal muscles in vivo using both local and systemic delivery.
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http://dx.doi.org/10.1007/978-1-59745-237-3_9DOI Listing
August 2008

Functionalized PEG-PEI copolymers complexed to exon-skipping oligonucleotides improve dystrophin expression in mdx mice.

Hum Gene Ther 2008 Aug;19(8):795-806

School of Biomedical Engineering, Drexel University, Philadelphia, PA 19104, USA.

Exon-skipping oligonucleotides (ESOs) with 2'-O-methyl modifications are promising compounds for the treatment of Duchenne muscular dystrophy (DMD). However, the usefulness of these compounds is limited by their poor delivery profile to muscle tissue in vivo. We previously established that copolymers made of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) enhanced ESO transfection in skeletal muscle of mdx mice, resulting in widespread distribution of dystrophin-positive fibers, but limited dystrophin expression by Western blot. In an attempt to improve ESO delivery and dystrophin expression, a new formulation of PEG-PEI copolymer was used, along with functionalized derivatives containing either the cell-penetrating peptide TAT (trans-activator of transcription), adsorbed colloidal gold (CG), or both TAT and CG. Tibialis anterior muscles were given three intramuscular injections of various PEG-PEI-ESO polyplexes (3 days apart; 5 microg of ESO per injection) and muscles were harvested 3 weeks after the first injection. Surface modifications of PEG-PEI copolymers with TAT showed the highest level of dystrophin recovery, with a 6-fold increase in dystrophin-positive fibers compared with ESO alone and up to 30% of normal dystrophin expression by Western blot. The adsorption of CG to either PEG-PEI or TAT-PEG-PEI copolymers showed no further improvement in dystrophin expression. Our data indicate that TAT-modified PEG-PEI copolymers are effective carriers for delivery of ESOs to skeletal muscle and are promising compounds for the therapeutic treatment of DMD.
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http://dx.doi.org/10.1089/hum.2007.129DOI Listing
August 2008

Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice.

BMC Biotechnol 2008 Apr 2;8:35. Epub 2008 Apr 2.

Drexel University College of Medicine, Department of Pharmacology and Physiology, Philadelphia, Pennsylvania 19102, USA.

Background: Exon skipping oligonucleotides (ESOs) of 2'O-Methyl (2'OMe) and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD). However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) are regarded as effective agents for enhanced delivery of nucleic acids in various applications.

Results: We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA) muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 microg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG) or adsorbtion of colloidal gold (CG), respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3-60 microg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal levels were found at 6 weeks after 10 twice-weekly injections of the NG-PEI2K-PEG550 copolymer complexed with 5 microg of ESO per injection. This injection and dosing regimen showed over 1000 dystrophin-positive fibers. H&E staining of all treated muscle groups revealed no overt signs of cytotoxicity.

Conclusion: We conclude that PEGylated PEI2K copolymers are efficient carriers for local delivery of 2'OMe ESOs and warrant further development as potential therapeutics for treatment of DMD.
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http://dx.doi.org/10.1186/1472-6750-8-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2362111PMC
April 2008

Induction of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers.

Mol Ther 2006 Jul 20;14(1):88-96. Epub 2006 Feb 20.

Department of Pharmacology and Physiology, Drexel University College of Medicine, Mailstop 488, NCB 8302, Philadelphia, PA 19102, USA.

Antisense oligonucleotides (AOs) with 2-O-methyl modifications can circumvent dystrophin mutations via exon skipping and, it is hoped, can become drugs for treatment of Duchenne muscular dystrophy (DMD). However, AO-based approaches are hindered by a lack of effective carriers to facilitate delivery of AOs to myonuclei. We examined whether copolymers composed of cationic poly(ethylene imine) (PEI) and polyethylene glycol (PEG) can enhance AO transfection in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEI2000(PEG550) copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no apparent cytotoxicity. Overall, injections of these low Mw polyplexes, which formed 250-nm aggregate particles, resulted in about sixfold more dystrophin-positive fibers than AO alone. Western analysis confirmed the dystrophin expression in these muscles. Surprisingly, injections of AO complexed with high Mw PEI25000(PEG5000) copolymers, which formed smaller nonaggregated particles, produced about threefold fewer dystrophin-positive fibers than injections of the low Mw polyplexes. We conclude that low Mw PEI2000(PEG550) copolymers function as high-capacity, nontoxic AO carriers suitable for in vivo transfection of skeletal muscle and are promising compounds for potential use in molecular therapy of DMD.
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http://dx.doi.org/10.1016/j.ymthe.2005.11.025DOI Listing
July 2006

Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides.

Biomacromolecules 2006 Jan;7(1):347-56

Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.
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http://dx.doi.org/10.1021/bm050726tDOI Listing
January 2006

Poly(ethylene imine)-poly(ethylene glycol) copolymers facilitate efficient delivery of antisense oligonucleotides to nuclei of mature muscle cells of mdx mice.

Hum Gene Ther 2005 Nov;16(11):1307-17

Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

Antisense oligonucleotides (AO) can facilitate dystrophin expression via targeted exon skipping in cultured cells of Duchenne muscular dystrophy (DMD) patients and in the mouse model of DMD (mdx mice). However, the lack of effective means to deliver AO to myonuclei remains the foremost limitation to their usefulness in DMD gene therapy. In this study we show that copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) facilitated efficient cellular uptake and nuclear delivery of AO in mature skeletal muscle fibers isolated from mdx mice. Confocal analysis of dual fluorescently tagged PEG-PEI-AO polyplexes, 24 hr after transfection, showed that the copolymer and AO were colocalized within punctate membrane- associated structures. Importantly, AO was efficiently translocated into myonuclei, whereas the copolymer was mostly excluded. The morphology of all transfected myofibers was perfectly maintained with no indication of damage or cytotoxicity. Quantitative fluorescence analysis showed that transfection with PEG-PEI-AO resulted in a 6-fold higher uptake of AO into myonuclei compared with transfections of AO alone. Interestingly, transfections with rhodamine-labeled PEG-PEI copolymers yielded an approximately 2- fold higher uptake of AO into myonuclei compared with transfections of unlabeled copolymers. Attempts to further increase AO delivery by addition of insulin-transferrin-selenium (ITS) to the medium showed no further improvement in AO delivery. Dose-response analysis indicated saturation of endocytotic uptake of the polyplex. Overall, we conclude that PEG-PEI copolymers represent high-capacity, nontoxic carriers for efficient delivery of AO to nuclei of mature myofibers.
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http://dx.doi.org/10.1089/hum.2005.16.1307DOI Listing
November 2005

Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens.

Am J Physiol Cell Physiol 2002 Apr;282(4):C835-44

Biomedical Sciences Graduate Group, Department of Orthopaedics, University of California, Veterans Affairs Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.

The myosin heavy chain (MHC) and myosin light chain (MLC) isoforms in skeletal muscle of Rana pipiens have been well characterized. We measured the force-velocity (F-V) properties of single intact fast-twitch fibers from R. pipiens that contained MHC types 1 or 2 (MHC1 or MHC2) or coexpressed MHC1 and MHC2 isoforms. Velocities were measured between two surface markers that spanned most of the fiber length. MHC and MLC isoform content was quantified after mechanics analysis by SDS-PAGE. Maximal shortening velocity (V(max)) and velocity at half-maximal tension (V(P 50)) increased with percentage of MHC1 (%MHC1). Maximal specific tension (P(o)/CSA, where P(o) is isometric tension and CSA is fiber cross-sectional area) and maximal mechanical power (W(max)) also increased with %MHC1. MHC concentration was not significantly correlated with %MHC1, indicating that the influence of %MHC1 on P(o)/CSA and W(max) was due to intrinsic differences between MHC isoforms and not to concentration. The MLC3-to-MLC1 ratio was not significantly correlated with V(max), V(P 50), P(o)/CSA, or W(max). These data demonstrate the powerful relationship between MHC isoforms and F-V properties of the two most common R. pipiens fiber types.
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http://dx.doi.org/10.1152/ajpcell.00482.2001DOI Listing
April 2002