Digital Drug Delivery: On-Off Ultrasound Controlled Antibiotic Release from Coated Matrices with Negligible Background Leaching.

Hydrogels, such as crosslinked poly(2-hydroxyethyl methacrylate) (pHEMA) have been used extensively in controlled release drug delivery systems. Our previous work demonstrated an ultrasound (US)-responsive system based on pHEMA coated with a self-assembled multilayer of C12-C18 methylene chains. The resulting coating was predominantly crystalline and relatively impermeable, forming an US-activated switch that controlled drug release on-demand, and kept the drug within the matrix in the absence of US. The device, as developed did, however, show a low background drug-leaching rate independent of US irradiation. For some applications, it is desirable to have very low or zero background release rates. This was achieved here by a combination of new processing steps, and by copolymerizing HEMA with a relatively hydrophobic monomer, hydroxypropyl methacrylate (HPMA). These advances produced systems with undetectable ciprofloxacin background release rates that are capable of US-facilitated drug release - up to 14-fold increases relative to controls both before and after US exposure. In addition, these observations are consistent with the hypothesis that US-mediated disorganization of the coating allows a transient flux of water into the matrix where its interaction with bound and dissolved drug facilitates its movement both within and out of the matrix.

Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery.

Ultrasound (US)-mediated gene delivery has emerged as a promising non-viral method for safe and selective gene delivery. When enhanced by the cavitation of microbubbles (MBs), US exposure can induce sonoporation that transiently increases cell membrane permeability for localized delivery of DNA. The present study explores the effect of generalizable MB customizations on MB facilitation of gene transfer compared to Definity®, a clinically available contrast agent. These modifications are 1) increased MB shell acyl chain length (RN18) for elevated stability and 2) addition of positive charge on MB (RC5K) for greater DNA associability. The MB types were compared in their ability to facilitate transfection of luciferase and GFP reporter plasmid DNA in vitro and in vivo under various conditions of US intensity, MB dosage, and pretreatment MB-DNA incubation. The results indicated that both RN18 and RC5K were more efficient than Definity®, and that the cationic RC5K can induce even greater transgene expression by increasing payload capacity with prior DNA incubation without compromising cell viability. These findings could be applied to enhance MB functions in a wide range of therapeutic US/MB gene and drug delivery approach. With further designs, MB customizations have the potential to advance this technology closer to clinical application.

Ultrasound-targeted microbubble destruction-mediated gene delivery into canine livers.

Ultrasound (US) was applied to a targeted canine liver lobe simultaneously with injection of plasmid DNA (pDNA)/microbubble (MB) complexes into a portal vein (PV) segmental branch and occlusion of the inferior vena cava (IVC) to facilitate DNA uptake. By using a 1.1 MHz, 13 mm diameter transducer, a fivefold increase in luciferase activity was obtained at 3.3 MPa peak negative pressure (PNP) in the treated lobe. For more effective treatment of large tissue volumes in canines, a planar unfocused transducer with a large effective beam diameter (52 mm) was specifically constructed. Its apodized dual element configuration greatly reduced the near-field transaxial pressure variations, resulting in a remarkably uniform field of US exposure for the treated tissues. Together with a 15 kW capacity US amplifier, a 692-fold increase of gene expression was achieved at 2.7 MPa. Transaminase and histology analysis indicated minimal tissue damage. These experiments represent an important developmental step toward US-mediated gene delivery in large animals and clinics.

Sustained antibiotic release from an intraocular lens-hydrogel assembly for cataract surgery.

PURPOSE: To develop a simple, novel polymeric drug-delivery device for prevention of postoperative bacterial infection after cataract surgery in the developing world. METHODS: A poly(2-hydroxyethyl-methacrylate) (pHEMA) hydrogel was developed to achieve sustained release characteristics of antibiotics. The in vitro antibiotic release kinetics and efficacy of antibiotic function were tested using a silicone biofilm model. In vivo feasibility was investigated using a rabbit model. The control group of rabbits underwent standard cataract surgery with intraocular lens (IOL) implant and postoperative topical antibiotic and steroid. The experimental group received the polymeric device inserted with standard three-piece IOL at the time of surgery and received only topical steroids postoperatively. In vivo intraocular antibiotic levels and outcomes after cataract surgery were evaluated. RESULTS: The in vitro studies demonstrate the antibiotic release kinetics can be controlled by optimization of the surface coating. The in vivo results showed sustained sufficient antibiotic concentration (above minimum inhibitory concentration for most common bacteria related to endophthalmitis) for >4 weeks. There was minimum toxicity observed in vivo. The device was effective in treating induced intraocular infection after cataract surgery. CONCLUSIONS: The initial findings of the polymeric drug-delivery device demonstrate the feasibility delivering sufficient antibiotic in the anterior chamber for the immediate postoperative period in a rabbit model. The device is simple to produce and may help alleviate the potential postsurgical infections in the developing nations.

Sustained release of antibiotic from poly(2-hydroxyethyl methacrylate) to prevent blinding infections after cataract surgery.

Intraocular lens implantation after opacified natural lens removal is the primary treatment for cataracts in developed countries. Cataract surgery is generally considered safe, but entails significant risks in countries where sophisticated sterile operating theaters are not widely available. Post-operative infection (endophthalmitis) is a potential blinding complication. Infection often results from bacterial colonization of the new lens implant and subsequent antibiotic-tolerant biofilm formation. To combat this risk, we developed a polymeric hydrogel system that can deliver effective levels of antibiotic over an extended period of time within the globe of the eye. Norfloxacin antibiotic was loaded into cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) gels, which were subsequently surface-modified with octadecyl isocyanate to produce a hydrophobic rate-limiting barrier controlling norfloxacin release. Octadecyl surface modification was characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). A 15-min modification leads to a uniform surface coating and near zero order release of norfloxacin from the matrix. Norfloxacin released from coated pHEMA kills Staphylococcus epidermidis in suspension and on a simulated medical implant surface. With these data, we demonstrate a new and effective system for sustained drug release from a hydrogel matrix with specific application for intraocular lens surgery.

Hemorrhage control in arteries using high-intensity focused ultrasound: a survival study.

High-intensity focused ultrasound (HIFU) has been shown to provide an effective method for hemorrhage control of blood vessels in acute animal studies. The objective of the current study was to investigate the long-term effects of HIFU-induced hemostasis in punctured arteries. The femoral arteries ( approximately 2mm in diameter) of 25 adult anesthetized rabbits were surgically exposed, and either punctured and treated with HIFU (n=15), served as control (no puncture and no HIFU application: n=7), or were punctured and left untreated (n=3). Treated animals were allowed to recover, and examined and/or sacrificed on days 0, 1, 3, 7, 14, 28, and 60 after treatment to obtain ultrasound images and samples of blood and tissue. Hemostasis (arrest of bleeding) was achieved in all 15 of the HIFU-treated arteries. Eleven of the arteries were patent after HIFU treatment, and four arteries were occluded, as determined by Doppler ultrasound. The median HIFU application time to achieve hemostasis was 20s (range 7-55 s) for the patent arteries and 110 s (range 50-134 s) for the occluded arteries. In untreated animals, bleeding had not stopped after 120 s. One of the occluded arteries had reopened by day 14. No immediate or delayed re-bleeding was observed after HIFU treatment. Maximal blood flow velocities were similar in HIFU-treated patent vessels and control vessels. No significant difference in hematocrits was found between HIFU-treated and control groups at different time points after the procedure. Light microscopy observations of the HIFU-treated arteries showed disorganization of adventitia, and coagulation and thinning of the tunica media. The general organization of the adventitia and tunica media recovered to normal appearance within 28 days, with some thinning of the tunica media observed up to day 60. Neointimal hyperplasia was observed on days 14 and 28. The results show that HIFU can produce effective and long-term (up to 60 days) hemostasis of punctured femoral arteries while preserving normal blood flow and vessel wall structure in the majority of vessels.

Gel phantom for use in high-intensity focused ultrasound dosimetry.

An optically transparent phantom was developed for use in high-intensity focused ultrasound (US), or HIFU, dosimetry studies. The phantom is composed of polyacrylamide hydrogel, embedded with bovine serum albumin (BSA) that becomes optically opaque when denatured. Acoustic and optical properties of the phantom were characterized as a function of BSA concentration and temperature. The speed of sound (1544 m/s) and acoustic impedance (1.6 MRayls) were similar to the values in soft tissue. The attenuation coefficient was approximately 8 times lower than that of soft tissues (0.02 Np/cm/MHz for 9% BSA). The nonlinear (B/A) coefficient was similar to the value in water. HIFU lesions were readily seen during formation in the phantom. In US B-mode images, the HIFU lesions were observed as hyperechoic regions only if the cavitation activity was present. The phantom can be used for fast characterization and calibration of US-image guided HIFU devices before animal or clinical studies.

Liver hemostasis with high-intensity ultrasound: repair and healing.

OBJECTIVE: Previous studies have shown that high-intensity focused ultrasound can effectively control bleeding from injuries of liver, spleen, and blood vessels. This study investigated long-term hemostasis and tissue repair after high-intensity focused ultrasound treatment in liver. METHODS: A total of 21 rabbits were randomly assigned to 2 groups: high-intensity focused ultrasound treatment (n = 14) and sham treatment (n = 7). All animals had sterile laparotomy and liver exposure. The high-intensity focused ultrasound-treated animals received liver incisions, 20 to 25 mm long and 4 to 6 mm deep, followed immediately by high-intensity focused ultrasound application until complete hemostasis was achieved. After recovery, sonographic images, blood samples, and histologic samples were collected immediately and on days 1, 3, 7, 14, 28, and 60 after treatment. RESULTS: All 14 liver injuries were hemostatic after an average +/- SD of 78 +/- 44 seconds of high-intensity focused ultrasound application, with no rebleeding at any time point after the treatment. Subsequent blood analysis showed no significant difference in serial hematologic or coagulation measures between the high-intensity focused ultrasound and sham groups. Alanine aminotransferase and aspartate aminotransferase levels increased immediately after surgery by as much as 285% up to day 3 and returned to normal values by day 7. Hematocrit and white blood cell counts showed no statistically significant difference from normal values at all time points. Histologic examination up to 60 days after treatment revealed scarring and liver tissue regeneration at the treatment site. CONCLUSIONS: High-intensity focused ultrasound appears to provide long-lasting hemostasis of acute liver injury. Healing and repair mechanisms after high-intensity focused ultrasound application appear to be intact.

Polyacrylamide gel as an acoustic coupling medium for focused ultrasound therapy.

A hydrogel acoustic coupling medium was investigated as a practical alternative to water for clinical applications of focused ultrasound (US) therapy. Material characterization and functional testing of polyacrylamide gel couplers were performed. Acoustic, bulk and thermal properties were measured. Conical couplers were designed and fabricated to fit a 3.5-MHz, spherically concave transducer for functional tests, including Schlieren imaging, power efficiency measurements and in vivo hemostasis experiments. Polyacrylamide was shown to have favorable acoustic properties that varied linearly with acrylamide concentration from 10% to 20% weight in volume. Attenuation coefficient, sound speed and impedance ranged from 0.08 to 0.14 dB/cm at 1 MHz, 1546 to 1595 m/s and 1.58 to 1.68 Mrayl, respectively. An intraoperative in vivo hemostasis experiment in a sheep model demonstrated that the gel-coupled transducer was capable of inducing hemostasis in actively bleeding splenic and hepatic incisions. The results of this study show that polyacrylamide may be a promising coupling material for focused US therapy.

Treatment of uterine fibroid tumors in an in situ rat model using high-intensity focused ultrasound.

OBJECTIVE: To determine the efficacy and safety of high-intensity focused ultrasound (HIFU) for the treatment of uterine fibroid tumors in an in situ animal model. DESIGN: High-intensity focused ultrasound was applied intraoperatively to uterine fibroid tumors in rats. SETTING: Department of Bioengineering, and Applied Physics Laboratory, University of Washington, Seattle, Washington. ANIMAL(S): Thirty-five tumors in 27 Eker rats that had spontaneous in situ uterine fibroids were randomly assigned into two groups receiving HIFU (n = 29) or sham (n = 6) treatments. INTERVENTION(S): Animals were anesthetized, and tumors were exposed surgically. The HIFU was applied at 3.5 MHz in 10-second bursts to produce coagulative necrosis lesions (3 mm by 10 mm), spaced 5 mm apart. Sham treatments consisted of exposing the tumors, and handling them similarly to those in the HIFU treatment group, but HIFU was not applied. MAIN OUTCOME MEASURE(S): Tumor volume was measured every week transabdominally using B-mode ultrasound imaging. Gross examination and histological analysis were performed after euthanasia. RESULT(S): More than half of the tumors in the HIFU treatment group showed significant tumor volume reduction. The average tumor volume in the sham treatment group increased 40-fold. Gross and histological analysis showed coagulative necrosis of tumor cells in the HIFU treatment group. CONCLUSION(S): The HIFU may provide an effective and safe method of treating uterine fibroid tumors.

High-intensity focused US: a potential new treatment for GI bleeding.

BACKGROUND: High-intensity focused US has been shown to achieve hemostasis in lacerated large veins and arteries. High-intensity focused US was studied as a potential endoscopic treatment for GI bleeding. METHODS: A segment of the auricular vein of the rabbit was lacerated longitudinally and then treated with a high-intensity focused US transducer driven at 3.9 MHz (focal intensity of 750 W/cm(2)) in 15 animals until hemostasis was achieved. Sham treatment was delivered to 3 vessels. Rabbits were euthanized on days 0, 2, 7, 14, and 28 to allow for histologic evaluation of the response to treatment. RESULTS: Hemostasis was achieved in all treated vessels and in none of the sham treatments. Mean treatment time was 13 seconds. Histology initially demonstrated acute thermal injury with subsequent thrombus formation and chronic inflammation leading to replacement of the vessel by fibrous scar tissue. CONCLUSIONS: High-intensity focused US causes hemostasis in acutely bleeding veins and results in occlusion of treated vessel with subsequent granulation tissue formation.

Spleen hemostasis using high-intensity ultrasound: survival and healing.

BACKGROUND: Previous studies have shown that high-intensity focused ultrasound (HIFU) can effectively control bleeding of incised livers and spleens and punctured vessels. This current study investigated the long-term safety of HIFU in splenic hemostasis. METHODS: A total of 21 rabbits were randomly assigned to two groups: HIFU treatment (n = 14), and sham treatment (n = 7). All animals underwent sterile laparotomy and splenic exposure. The HIFU-treated animals received splenic incisions, 8 to 10 mm long and 4 to 5 mm deep, and immediate 9.6-MHz HIFU until hemostasis was achieved. After recovery, ultrasound images, blood samples, and histologic samples were collected on days 0, 1, 3, 7, 14, 28, and 60. RESULTS: All 14 splenic injuries were hemostatic after an average of 96 seconds of HIFU application. There was evidence of rebleeding in one animal between days 3 and 7 posttreatment. Subsequent blood analysis showed no significant difference in serial hematologic or coagulation measures between HIFU and sham groups. Histologic examination up to 60 days posttreatment revealed scarring and spleen tissue regeneration at the treatment site. CONCLUSION: HIFU provides an effective and safe method of achieving hemostasis after acute splenic injury.

Treatment of uterine leiomyosarcoma in a xenograft nude mouse model using high-intensity focused ultrasound: a potential treatment modality for recurrent pelvic disease.

OBJECTIVE: The objective was to test the efficacy of high-intensity focused ultrasound (HIFU) for treatment of uterine leiomyosarcoma in a Xenograft nude mouse model. METHODS: A total of 65 athymic nude mice were inoculated subcutaneously with 5 to 7 x 10(6) ELT-5B cells, a uterine leiomyosarcoma cell line derived from the Eker rat. Thirty animals showed tumor growth. The tumor volume was measured transcutaneously once a week. Animals were randomly assigned to three groups: HIFU treatment (n = 17), sham treatment (n = 7), and control (n = 6). A HIFU device, operating at a frequency of 2.0 MHz and an intensity of 2000 W/cm(2), was used for treatment. RESULTS: Within 3 weeks of a single HIFU treatment, 100% reduction in tumor volume was observed in all animals, except one. A second HIFU treatment was applied to that animal, resulting in 100% reduction in tumor volume. The tumors in the sham-treated animals continued to grow at a similar rate to that of the control group to approximately 500% of the tumor volume at the time of treatment. All animals were monitored for a maximum of 3 months. No metastasis was observed in the HIFU-treated animals. Histological examination confirmed a complete tumor disappearance after HIFU treatment. CONCLUSION: We have shown that HIFU can effectively treat uterine leiomyosarcoma tumors inoculated in Xenograft nude mice, demonstrating HIFU's potential use for treatment of recurrent uterine leiomyosarcoma.