Biomedical subjects

Margaret A Wheatley

Publications and source records attributed to Margaret A Wheatley.

10 recordsLinked to original sources

PubMed2006

Comparison of in vitro and in vivo acoustic response of a novel 50:50 PLGA contrast agent.

A comparison between in vitro and in vivo experiments conducted to investigate the acoustic properties of a novel, 1.2 microm diameter poly(lactic-co-glycolic acid) (50:50) (PLGA) ultrasound contrast agent, the development of which was described previously by us, is presented. A pulse-echo setup was used to determine enhancement in vitro. Additional in vitro studies further characterized the hollow microcapsules, including resonance frequency from attenuation measurements (from 2.25 to 15 MHz) and temperature effects (25 degrees C vs. 37 degrees C). In vivo, four rabbits received intravenous injections of the agent (dose range: 0.005-0.13 ml/kg). Quantitative in vivo dose-responses were calculated off-line using spectral power analysis of audio Doppler signals acquired from a custom-made 10 MHz cuff transducer placed around the surgically exposed distal aorta. This frequency was chosen since the very shallow scanning depths encountered in rabbits, in particular for the cuff transducer placed directly around the vessel, necessitates the use of high frequency imaging devices with sufficient spatial resolution to enable meaningful measurements. For qualitative assessments, two rabbits were imaged pre- and post-contrast administration (dose: 0.1 ml/kg) in power Doppler mode. Significant acoustic enhancements (up to 24 dB) were reported both in vitro and in vivo. Moreover, the rabbits did not show any adverse side effects from multiple injections (>20) of the agent. Measured in vitro resonance frequency between 3.09 and 3.49 MHz was lower than predicted for a similar sized free bubble, potentially due to capsule wall structure. Minimal loss of signal (approximately 4 dB) was observed at 25 degrees C over 20 min of insonation at 5 MHz but at 37 degrees C the signal dropped close to base line within the first 5 min. This temperature sensitivity could be due to loss of capsule integrity (and hence loss of gas). Potential causes include increased hydrolysis or polymer softening and increased water uptake by the shell at temperatures closer to the glass transition temperature (T(g)).

Acoustics↗

PubMed2006

Surfactant-stabilized contrast agent on the nanoscale for diagnostic ultrasound imaging.

Ultrasound contrast agents (CA) are generally micron-sized stabilized gas bubbles, injected IV. However, to penetrate beyond the vasculature and accumulate in targets such as tumors, CA must be an order of magnitude smaller. We describe a method of achieving nanometer-sized, surfactant-stabilized CA by differential centrifugation. High g force was shown to destroy bubble integrity. Optimal conditions (300 rpm for 3 min) produced an agent with a mean diameter of 450 nm, which gave 25.5 dB enhancement in vitro at a dose of 10 microL/mL, with a 13 min half-life. In vivo, the CA produced excellent power Doppler and grey-scale pulse inversion harmonic images at low acoustic power when administered. In vivo dose-response curves obtained in three rabbits showed enhancement between 20 and 25 dB for dosages above 0.025 mL/kg. These results encourage further investigation of the possible diagnostic and therapeutic benefits of using nanoparticles as CA, including passive targeting and accumulation in tumors.

Animals↗

PubMed2005

Alginate encapsulated BDNF-producing fibroblast grafts permit recovery of function after spinal cord injury in the absence of immune suppression.

Encapsulation of cells has the potential to provide a protective barrier against host immune cell interactions after grafting. Previously we have shown that alginate encapsulated BDNF-producing fibroblasts (Fb/BDNF) survived for one month in culture, made bioactive neurotrophins, survived transplantation into the injured spinal cord in the absence of immune suppression, and provided a permissive environment for host axon growth. We extend these studies by examining the effects of grafting encapsulated Fb/BDNF into a subtotal cervical hemisection on recovery of forelimb and hindlimb function and axonal growth in the absence of immune suppression. Grafting of encapsulated Fb/BDNF resulted in partial recovery of forelimb usage in a test of vertical exploration and of hindlimb function while crossing a horizontal rope. Recovery was significantly greater compared to animals that received unencapsulated Fb/BDNF without immune suppression, but similar to that of immune suppressed animals receiving unencapsulated Fb/BDNF. Immunocytochemical examination revealed neurofilament (RT-97), 5-HT, CGRP and GAP-43 containing axons surrounding encapsulated Fb/BDNF within the injury site, indicating axonal growth. BDA labeling however showed no evidence of regeneration of rubrospinal axons in recipients of encapsulated Fb/BDNF, presumably because the amounts of BDNF available from the encapsulated grafts are substantially less than those provided by the much larger numbers of Fb/BDNF grafted in a gelfoam matrix in the presence of immune suppression. These results suggest that plasticity elicited by the BDNF released from the encapsulated cells contributed to reorganization that led to behavioral recovery in these animals and that the behavioral recovery could proceed in the absence of rubrospinal tract regeneration. Alginate encapsulation is therefore a feasible strategy for delivery of therapeutic products produced by non-autologous engineered fibroblasts and provides an environment suitable for recovery of lost function in the injured spinal cord.

Alginates↗

PubMed2004

Peptide-modified alginate surfaces as a growth permissive substrate for neurite outgrowth.

Different strategies are being investigated for treatment of spinal cord injuries, one of the most promising being application of neurotrophic factors, which have been shown to prevent neuronal death and stimulate regeneration of injured axons. Ex vivo gene therapy has emerged as the leading delivery method at the site of the injury, and we have shown previously that encapsulating genetically engineered fibroblasts in an immunoprotective alginate capsule can permit implantation of the factor-secreting cells without need for immunosuppression. This strategy could be greatly enhanced by providing the sprouting neurons with a permissive substrate upon which to attach and grow. We report here studies on the modification of an alginate gel surface by either coating it with laminin or by covalent attachment of YIGSR peptide. Using NB2a neuroblastoma cells, we found that native alginate elicited minimal cell attachment ( approximately 1.5%); however, YIGSR-alginate conjugate elicited a fivefold increase in numbers of cells attached using peptide ratios of 0.5 and 1 mg/g alginate, ranging from 9.5% of the cells at the lower ratio, to about 44% at the higher. Only a further 19% increase was obtained at an increased peptide density of 2 mg/g alginate ( approximately 63% over control). Laminin-coated gels showed approximately 60% cell attachment. However, laminin coating did not stimulate differentiation and neurite growth, whereas both numbers and lengths of outgrowths increased with increasing peptide density on peptide-modified alginate. We demonstrate here the ability of the peptide-modified alginate gels to allow adhesion of NB2a neuroblastoma cells and to promote neurite outgrowth from these cells when attached to the peptide-modified alginate surface. Also, we show that the adhesion of NB2a neuroblastoma cells and neurite outgrowth from the attached cells is a function of the peptide density on the gel surface.

Alginates↗

PubMed2004

Ultrasound degradation of novel polymer contrast agents.

This report describes an investigation into factors affecting the degradation of novel poly(lactic-co-glycolic acid) (PLGA) contrast agents. Contrast agents fabricated by two different methods and varying in acoustic properties were compared. The effect of ultrasound frequency (5 and 10 MHz) on degradation of the microcapsules was also studied. High-performance liquid chromatography was used to quantify the production of lactic and glycolic acid to monitor agent degradation. The degradation pattern from the microcapsules was found to be closely related to capsule morphology; the more acoustically efficient capsules (maximum enhancement of 25 dB at 5 MHz with 0.004 mg/mL) degraded at a faster rate than those with lower acoustical efficiency (maximum enhancement of 25 dB at 5 MHz only achieved with 0.6 mg/mL). The capsules also degraded fastest when insonated at the frequency at which they gave highest backscatter. In addition, despite the use of a 50:50 PLGA copolymer, more glycolic than lactic acid was released at early time points, which reflects the greater hydrophilicity of the glycolic acid residues, and greater degradation rate of glycolic acid repeat units. The results from this study provided unique insight into the degradation behavior of hollow PLGA microcapsules, and their potential in ultrasound diagnosis and therapy.

Biocompatible Materials↗

PubMed2004

Development and characterization of a nano-scale contrast agent.

Agents injected parenterally must be less than approximately 8 microm diameter in order to traverse the capillaries in the pulmonary bed, but these agents remain in the vasculature until they are eliminated from the body by a variety of mechanisms. Targeting of cells outside the capillaries requires agent diameters of less than approximately 700 nm to enable escape through the larger-than-usual pores that have been noted in the leaky vasculature of a tumor. The objective of this study was to test the feasibility of creating a surfactant-stabilized nano-bubble with favorable acoustic properties, and identify the key parameters that influence size, yield and stability. Size distribution was characterized using laser light scattering. In vitro acoustic enhancement was assessed by generation of dose and time response curves. We previously developed a successful protocol to generate gas-filled microbubbles (containing perfluorocarbon, sulfur hexafluoride or air) with mean diameter of 1.5 microm, using sonication of carefully selected surfactant mixtures. This presentation describes generation of nano-bubbles with mean diameters ranging from 700 to 450 nm, depending on process variables. In all cases a centrifugation step was employed to separate the nano-sized particles. The in vitro dose response curves gave a maximum of 23-27 dB enhancement compared to buffer in the absence of agent, with the maximum enhancement and presence of shadowing at higher doses being dependent on the fabrication protocol. The effect of sonication time for solutions containing a mixture of the surfactants (Span 60 and Tween 80) was also tested, but was determined not to be an influencing factor. Future studies will involve development of a mathematical model characterizing the mean size as a function of centrifugal force, spin time and initial size distribution. Future work will also include imaging of tumor-bearing mice and measuring imaging potential in vivo in New Zealand white rabbits using power Doppler.

Contrast Media↗

PubMed2004

Polymeric contrast agent with targeting potential.

The Arg-Gly-Asp (RGD) peptide sequence was conjugated to poly (lactid acid), (PLA), microcapsules. These hollow, biodegradable PLGA microcapsules were developed in our laboratory for use as ultrasound contrast agents. By modifying the surface of the agent with a targeting ligand, it can be targeted to a specific address within the body. This application is ideal for both targeted imaging and/or targeted drug delivery. Integrins are membrane-spanning proteins in cells that play a vital role in cell attachment and many other processes. The RGD peptide sequence targets integrins expressed during angiogenesis, alphavbeta3 and alphavbeta5. The integrins specific to angiogenesis are more active during cancer and can be used as receptors for the RGD-conjugated contrast agents. Although the generic RGD sequence is not specific to only alphavbeta3 and alphavbeta5 integrins, it is an excellent candidate for proof of concepts studies such as described here. Preliminary in vitro results indicate that the modified capsules remain highly echogenic (maximum enhancement of 20 dB in vitro) and adhere specifically to a breast cancer cell line MDA-MB-231 in static experiments. However, no adherence is seen with either unmodified capsules (negative control), or when cells that have been pre-saturated with RGD ligand are contacted with modified capsules (positive control). Specific targeting of ultrasound contrast agents could lead the way to imaging as a method for discrimination of malignant from benign.

Analysis of Variance↗

PubMed2004

Effect of shell type on the in vivo backscatter from polymer-encapsulated microbubbles.

This study compared in vivo enhancement from four different polymer-encapsulated ultrasound (US) contrast agents. The agents were produced with a rigid shell composed of the biodegradable block copolymer poly[D,L-lactide-co-glycolide] (PLGA) with the lactic and glycolic acid ratios 50:50, 75:25, 85:15 and 100:0 (i.e., increasingly hydrophobic shell compositions). Approximately the same bubble diameter (1.2 microm) and concentration (0.4 g/mL) were obtained for each agent. In four rabbits, audio Doppler signals were acquired from a 10 MHz cuff transducer placed around a surgically exposed vessel (contrast dose: 0.0125 to 0.15 mL/kg). In vivo dose responses were calculated off-line (in dB). Nine rabbit kidneys were imaged during contrast administration (0.1 mL/kg) in power Doppler and grey-scale pulse inversion harmonic (PIHI) modes using an HDI 5000 scanner (Philips Medical Systems, Bothell, WA). Time-intensity curves were produced and the time-to-peak, peak intensity, slope, area under the curve (AUC) and total duration of enhancement for each agent were compared. All agents produced marked Doppler enhancement with increasing duration from the 50:50 agent (48 +/- 10 s) to the 75:25 agent (166 +/- 46 s), the 85:15 agent (403 +/- 83 s) and with the 100:0 agent (603 +/- 93 s) lasting longest (p < 0.02). No other parameters changed significantly, except the AUC of the 85:15 agent, which was greater than that of the 50:50 agent (190.75 vs. 61.58; p = 0.02). The in vivo dose-response curves were similar for all agents, with mean enhancement up to 20.6 +/- 1.11 dB (p = 0.17). In conclusion, contrast duration increases by an order of magnitude as the lactic acid component in the polymer-encapsulated bubbles increases and the shell, thus, becomes increasingly hydrophobic.

Animals↗

PubMed2003

Development of a novel method for synthesis of a polymeric ultrasound contrast agent.

Medical ultrasound is a highly valuable diagnostic tool, especially when compared with other imaging modalities. It is a noninvasive, real-time, portable, extremely safe method compared with X-ray and inexpensive relative to magnetic resonance imaging. However, ultrasound is limited in its ability to distinguish between diseased and normal tissue. This limitation has led to the development of contrast agents. We have produced novel poly (lactic-co-glycolic) acid air-filled microcapsules that work well as ultrasound contrast agents, giving up to 24 and 25 dB enhancement when insonated in the medical imaging range at 5 and 7.5 MHz, respectively. The capsules were fabricated by modifying a double emulsion method to encapsulate camphor in the oil phase and ammonium carbonate in the aqueous phase, and later sublime the encapsulated material, leaving voids capable of being filled with a gas in their place. The role of the surfactant, poly vinyl alcohol, solution temperature, was studied and found to play an important role in the morphology of the capsules, altering their acoustic response.

Dose-Response Relationship, Drug↗

PubMed2003

Microencapsulated liposomes in controlled drug delivery: strategies to modulate drug release and eliminate the burst effect.

The release of fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) from alginate-microencapsulated liposomes was studied to evaluate the properties of this system for controlled drug delivery. Liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) (molar ratio 7:3) and of PC, phosphatidylglycerol (PG), and cholesterol (6:1:3) were encapsulated in alginate (Alg) crosslinked with Ca(2+) (Ca-Alg), Al(3+) (Al-Alg), and Ba(2+) (Ba-Alg). Capsules were coated with poly(l-ornithine) followed by a final alginate coat. A rapid initial burst of protein release was observed from liposomes encapsulated in Ca-Alg and Al-Alg. No burst was observed when liposomes were encapsulated in Ba-Alg, indicating that the crosslinking ions could significantly affect the release of entrapped protein. Also, the release from encapsulated liposomes varied significantly with liposome composition, especially with Ca-Alg as observed with encapsulation of PC, dioleoylphosphatidylcholine (DOPC), and DOPC/Chol liposomes. Cholesterol increased the leakiness of the liposomes after encapsulation. In all cases, the release from microencapsulated liposomes was much faster than that from free liposomes suggesting an interaction between the liposomes and the alginate. Differential scanning calorimetry supports the hypothesis that alginate was inserted into the lipid bilayer resulting in a rapid release of protein from microencapsulated liposomes. Moreover, it was observed that the degree of interaction between liposomes and alginate varied with liposome composition.

Delayed-Action Preparations↗