Nanoparticle-Mediated Histotripsy Using Dual-Frequency Pulsing Methods.

OBJECTIVE: Nanoparticle-mediated histotripsy (NMH) is a novel ablation method that combines nanoparticles as artificial cavitation nuclei with focused ultrasound pulsing to achieve targeted, non-invasive, and cell-selective tumor ablation. The study described here examined the effect of dual-frequency histotripsy pulsing on the cavitation threshold, bubble cloud characteristics, and ablative efficiency in NMH. High-speed optical imaging was used to analyze bubble cloud characteristics and to measure ablation efficiency for NMH inside agarose tissue phantoms containing perfluorohexane-filled nanocone clusters, which were previously developed to reduce the histotripsy cavitation threshold for NMH. METHODS: Dual-frequency histotripsy pulsing was applied at a 1:1 pressure ratio using a modular 500 kHz and 3 MHz dual-frequency array transducer. Optical imaging results revealed predictable, well-defined bubble clouds generated for all tested cases with similar reductions in the cavitation thresholds observed for single-frequency and dual-frequency pulsing. RESULTS: Dual-frequency pulsing was seen to nucleate small, dense clouds in agarose phantoms, intermediate in size of their component frequencies but closer in area to that of the higher component frequency. Red blood cell experiments revealed complete ablations were generated by dual-frequency NMH in all phantoms in <1500 pulses. This result was a significant increase in ablation efficiency compared with the ∼4000 pulses required in prior single-frequency NMH studies. CONCLUSION: Overall, this study indicates the potential for using dual-frequency histotripsy methods to increase the ablation efficacy of NMH.

Particle-Mediated Histotripsy for the Targeted Treatment of Intraluminal Biofilms in Catheter-Based Medical Devices.

Objective. This paper is an initial work towards developing particle-mediated histotripsy (PMH) as a novel method of treating catheter-based medical device (CBMD) intraluminal biofilms. Impact Statement. CBMDs commonly become infected with bacterial biofilms leading to medical device failure, infection, and adverse patient outcomes. Introduction. Histotripsy is a noninvasive focused ultrasound ablation method that was recently proposed as a novel method to remove intraluminal biofilms. Here, we explore the potential of combining histotripsy with acoustically active particles to develop a PMH approach that can noninvasively remove biofilms without the need for high acoustic pressures or real-time image guidance for targeting. Methods. Histotripsy cavitation thresholds in catheters containing either gas-filled microbubbles (MBs) or fluid-filled nanocones (NCs) were determined. The ability of these particles to sustain cavitation over multiple ultrasound pulses was tested after a series of histotripsy exposures. Next, the ability of PMH to generate selective intraluminal cavitation without generating extraluminal cavitation was tested. Finally, the biofilm ablation and bactericidal capabilities of PMH were tested using both MBs and NCs. Results. PMH significantly reduced the histotripsy cavitation threshold, allowing for selective luminal cavitation for both MBs and NCs. Results further showed PMH successfully removed intraluminal biofilms in Tygon catheters. Finally, results from bactericidal experiments showed minimal reduction in bacteria viability. Conclusion. The results of this study demonstrate the potential for PMH to provide a new modality for removing bacterial biofilms from CBMDs and suggest that additional work is warranted to develop histotripsy and PMH for treatment of CBMD intraluminal biofilms.

Bubble Cloud Behavior and Ablation Capacity for Histotripsy Generated from Intrinsic or Artificial Cavitation Nuclei.

The study described here examined the effects of cavitation nuclei characteristics on histotripsy. High-speed optical imaging was used to compare bubble cloud behavior and ablation capacity for histotripsy generated from intrinsic and artificial cavitation nuclei (gas-filled microbubbles, fluid-filled nanocones). Results showed a significant decrease in the cavitation threshold for microbubbles and nanocones compared with intrinsic-nuclei controls, with predictable and well-defined bubble clouds generated in all cases. Red blood cell experiments showed complete ablations for intrinsic and nanocone phantoms, but only partial ablation in microbubble phantoms. Results also revealed a lower rate of ablation in artificial-nuclei phantoms because of reduced bubble expansion (and corresponding decreases in stress and strain). Overall, this study demonstrates the potential of using artificial nuclei to reduce the histotripsy cavitation threshold while highlighting differences in the bubble cloud behavior and ablation capacity that need to be considered in the future development of these approaches.

Nanoparticle-mediated histotripsy (NMH) using perfluorohexane 'nanocones'.

Nanoparticle-mediated histotripsy (NMH) is an ultrasound treatment strategy that combines acoustically sensitive nanoparticles with histotripsy. Previous NMH studies using perfluorocarbon (PFC) nanodroplets (ND's), ~200 nm in diameter, demonstrated that NMH can selectively generate cavitation by reducing the cavitation threshold from ~25-30 MPa to ~10-15 MPa. Recent studies have also shown that cavitation nucleation in NMH is directly caused by the incident negative pressure (p-) exposed to the PFC, as predicted by classical nucleation theory (CNT), suggesting that the NMH cavitation threshold is dependent on the total volume of PFC present in the focal region. In this study, we investigate the use of a newly developed NMH nanoparticle synthesized using an inclusion complex of methylated ß-cyclodextrin and perfluorohexane (PFH). These 'nanocones' (NCs) have advantages compared to previously used ND's due to their smaller size (~50 nm), simple synthesis method, higher stability and information of definite PFH amount carried by the NC. To test the hypothesis that NCs can reduce the NMH cavitation threshold similar to ND's, and that the NMH cavitation threshold is dependent upon the total PFH concentration, tissue phantoms containing concentrations of NCs ranging from 10-5 to 10-10 (ml PFH/ml water) were exposed to single cycle ultrasound pulses using a 500 kHz focused transducer where high speed imaging captured cavitation data. Results showed that NCs significantly reduced the histotripsy cavitation threshold to 11.0 MPa for a concentration of 10-5 (ml PFH/ml water), with the threshold increasing at lower concentrations. Finally, the ability of NCs to be used for effective NMH ablation was demonstrated in tissue phantoms containing red blood cells (RBCs). Overall, the results of the study support our hypotheses that NCs can be used for effective NMH therapy and that NC concentration has a predictable threshold-reducing effect.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on "smart" particles.

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming "smart" nanoparticles. Using ß-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that "smart" nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.

Effects of Droplet Composition on Nanodroplet-Mediated Histotripsy.

Nanodroplet-mediated histotripsy (NMH) is a targeted ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. In two previous studies, polymer-encapsulated perfluoropentane nanodroplets were used to generate well-defined ablation similar to that obtained with histotripsy, but at significantly lower pressure, when NMH therapy was applied at a pulse repetition frequency (PRF) of 10 Hz. However, cavitation was not maintained over multiple pulses when ultrasound was applied at a lower PRF (i.e., 1-5 Hz). We hypothesized that nanodroplets with a higher-boiling-point perfluorocarbon core would provide sustainable cavitation nuclei, allowing cavitation to be maintained over multiple pulses, even at low PRF, which is needed for efficient and complete tissue fractionation via histotripsy. To test this hypothesis, we investigated the effects of droplet composition on NMH therapy by applying histotripsy at various frequencies (345 kHz, 500 kHz, 1.5 MHz, 3 MHz) to tissue phantoms containing perfluoropentane (PFP, boiling point ∼29°C, surface tension ∼9.5 mN/m) and perfluorohexane (PFH, boiling point ∼56°C, surface tension ∼11.9 mN/m) nanodroplets. First, the effects of droplet composition on the NMH cavitation threshold were investigated, with results revealing a significant decrease (>10 MPa) in the peak negative pressure (p-) cavitation threshold for both types of nanodroplets compared with controls. A slight decrease (∼1-3 MPa) in threshold was observed for PFP phantoms compared with PFH phantoms. Next, the ability of nanodroplets to function as sustainable cavitation nuclei over multiple pulses was investigated, with results revealing that PFH nanodroplets were sustainable cavitation nuclei over 1,000 pulses, whereas PFP nanodroplets were destroyed during the first few pulses (<50 pulses), likely because of the lower boiling point. Finally, tissue phantoms containing a layer of embedded red blood cells were used to compare the damage generated for NMH treatments using PFP and PFH droplets, with results indicating that PFH nanodroplets significantly improved NMH ablation, allowing for well-defined lesions to be generated at all frequencies and PRFs tested. Overall, the results of this study provide significant insight into the role of droplet composition in NMH therapy and provide a rational basis to tailor droplet parameters to improve NMH tissue fractionation.

The role of positive and negative pressure on cavitation nucleation in nanodroplet-mediated histotripsy.

Nanodroplet-mediated histotripsy (NMH) is an ultrasound ablation technique combining histotripsy with acoustically sensitive perfluorocarbon (PFC) nanodroplets that can be selectively delivered to tumor cells for targeted tumor ablation. NMH takes advantage of the significantly reduced cavitation threshold of the nanodroplets, allowing for cavitation to be selectively generated only in regions containing nanodroplets. Understanding the physical mechanisms underlying the nanodroplet cavitation process is essential to the development of NMH. In this study, we hypothesize that cavitation nucleation is caused by the negative pressure (p-) exposed to the PFC, and the NMH cavitation threshold is therefore determined by the incident p- of the single-cycle pulses commonly used in NMH. This paper reports the first study that separately investigates the effects of negative and positive pressure on the NMH cavitation threshold using near half-cycle ultrasound pulses with dominant negative (negative-polarity pulses) or positive (positive-polarity pulses) pressure phases. Tissue phantoms containing perfluorohexane (PFH) nanodroplets were exposed to negative-polarity and positive-polarity pulses generated by a frequency compounding transducer recently developed in our lab, and the probability of generating cavitation was measured as a function of peak negative (p-) and peak positive (p+) pressure. The results showed close agreement in the p- cavitation threshold for PFH phantoms exposed to negative-polarity (11.4 ± 0.1 MPa) and positive-polarity (11.7 ± 0.2 MPa) pulses. The p+ at the cavitation threshold, in contrast, was measured to be sign ficantly different for the negative-polarity (4.0 ± 0.1 MPa) and positive-polarity (42.6 ± 0.2 MPa) pulses. In the final part of this study, the experimental results were compared to the cavitation threshold predicted by classical nucleation theory (CNT), with results showing close agreement between simulations and experiments. Overall, the results support our hypothesis and provide significant insight into the physical mechanisms underlying NMH.

"Smart" Nanoparticles Enhance the Cytoplasmic Delivery of Anti-RhoC Silencing RNA and Inhibit the Migration and Invasion of Aggressive Breast Cancer Cells.

Rho-GTPases are small GTP-binding proteins that contribute to the epithelial-to-mesenchymal transition by regulating several cellular processes including organization of the actin cytoskeleton, cell motility, transcription, and cell proliferation. Overexpression of RhoC-GTPases (RhoC) in breast cancer has been implicated in poor disease prognosis due to increased cancer cells invasion, migration, and motility, which warranted its consideration as a therapeutic target for inhibiting breast cancer metastasis. Using silencing RNA (siRNA) molecules to knockdown RhoC expression is a promising approach to inhibit breast cancer metastases. However, transforming anti-RhoC siRNA molecules into a viable therapy remains a challenge due to the lack of a biocompatible carrier that can selectively deliver the RNA cargo into breast cancer cells. We report the use of a degradable, pH-sensitive, ß-cyclodextrin (ßCD)-based polymeric carrier that condenses anti-RhoC siRNA forming "smart" particles. These smart anti-RhoC particles were efficiently internalized, successfully escaped the endosome, and delivered the RNA cargo into the cytoplasm of SUM149 and MDA-MB-231 breast cancer cells. Our results show that anti-RhoC particles used at a low N/P ratio of 2.5/1 suppressed RhoC protein levels by 100% and 90% in SUM149 and MDA-MB-231 cells, respectively. Further, anti-RhoC particles inhibited the invasion, motility, and migration of SUM149 and MDA-MB-231 cells by 40-47%, 57-60%, and 61.5-73%, respectively. Smart particles encapsulating the scrambled siRNA sequence did not affect RhoC protein expression or the invasion, motility, and migration of SUM149 and MDA-MB-231 cells, which indicate the biocompatibility of the polymeric carrier and selectivity of the observed RhoC knockdown. These results collectively indicate the therapeutic potential of smart anti-RhoC particles in arresting the metastatic spread of breast cancer cells.

Nanodroplet-mediated histotripsy for image-guided targeted ultrasound cell ablation.

This paper is an initial work towards developing an image-guided, targeted ultrasound ablation technique by combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. Using extremely short, high-pressure pulses, histotripsy generates a dense cloud of cavitating microbubbles that fractionates tissue. We hypothesize that synthetic nanodroplets that encapsulate a perfluoropentane (PFP) core will transition upon exposure to ultrasound pulses into gas microbubbles, which will rapidly expand and collapse resulting in disruption of cells similar to the histotripsy process but at a significantly lower acoustic pressure. The significantly reduced cavitation threshold will allow histotripsy to be selectively delivered to the tumor tissue and greatly enhance the treatment efficiency while sparing neighboring healthy tissue. To test our hypothesis, we prepared nanodroplets with an average diameter of 204 ± 4.7 nm at 37°C by self-assembly of an amphiphilic triblock copolymer around a PFP core followed by cross-linkage of the polymer shell forming stable nanodroplets. The nanodroplets were embedded in agarose tissue phantoms containing a sheet of red blood cells (RBCs), which were exposed to 2-cycle pulses applied by a 500 kHz focused transducer. Using a high speed camera to monitor microbubble generation, the peak negative pressure threshold needed to generate bubbles >50 µm in agarose phantoms containing nanodroplets was measured to be 10.8 MPa, which is significantly lower than the 28.8 MPa observed using ultrasound pulses alone. High speed images also showed cavitation microbubbles produced from the nanodroplets displayed expansion and collapse similar to histotripsy alone at higher pressures. Nanodroplet-mediated histotripsy created consistent, well-defined fractionation of the RBCs in agarose tissue phantoms at 10 Hz pulse repetition frequency similar to the lesions generated by histotripsy alone but at a significantly lower pressure. These results support our hypothesis and demonstrate the potential of using nanodroplet-mediated histotripsy for targeted cell ablation.

Targeting hepatic cancer cells with pegylated dendrimers displaying N-acetylgalactosamine and SP94 peptide ligands.

Poly(amidoamine) (PAMAM) dendrimers are branched water-soluble polymers defined by consecutive generation numbers (Gn) indicating a parallel increase in size, molecular weight, and number of surface groups available for conjugation of bioactive agents. In this article, we compare the biodistribution of N-acetylgalactosamine (NAcGal)-targeted [(14) C]1 -G5-(NH2 )5 -(Ac)108 -(NAcGal)14 particles to non-targeted [(14) C]1 -G5-(NH2 )127 and PEGylated [(14) C]1 -G5-(NH2 )44 -(Ac)73 -(PEG)10 particles in a mouse hepatic cancer model. Results show that both NAcGal-targeted and non-targeted particles are rapidly cleared from the systemic circulation with high distribution to the liver. However, NAcGal-targeted particles exhibited 2.5-fold higher accumulation in tumor tissue compared to non-targeted ones. In comparison, PEGylated particles showed a 16-fold increase in plasma residence time and a 5-fold reduction in liver accumulation. These results motivated us to engineer new PEGylated G5 particles with PEG chains anchored to the G5 surface via acid-labile cis-aconityl linkages where the free PEG tips are functionalized with NAcGal or SP94 peptide to investigate their potential as targeting ligands for hepatic cancer cells as a function of sugar conformation (α versus ß), ligand concentration (100-4000 nM), and incubation time (2 and 24 hours) compared to fluorescently (Fl)-labeled and non-targeted G5-(Fl)6 -(NH2 )122 and G5-(Fl)6 -(Ac)107 -(cPEG)15 particles. Results show G5-(Fl)6 -(Ac)107 -(cPEG[NAcGalß ])14 particles achieve faster uptake and higher intracellular concentrations in HepG2 cancer cells compared to other G5 particles while escaping the non-specific adsorption of serum protein and phagocytosis by Kupffer cells, which make these particles the ideal carrier for selective drug delivery into hepatic cancer cells.

Enzyme-activated nanoconjugates for tunable release of doxorubicin in hepatic cancer cells.

We report the synthesis of a series of aromatic azo-linkers (L1-L4), which are selectively recognized and cleaved by azoreductase enzymes present in the cytoplasm of hepatic cancer cells via a NADPH-dependent mechanism. We utilized L1-L4 azo-linkers to conjugate doxorubicin to generation 5 (G5) of poly(amidoamine) dendrimers to prepare G5-L(x)-DOX nanoconjugates. We incorporated electron-donating oxygen (O) or nitrogen (N) groups in the para and ortho positions of L1-L4 azo-linkers to control the electronegativity of G5-L(x)-DOX conjugates and investigated their cleavage by azoreductase enzymes and the associated release of loaded DOX molecules. Hammett σ values of G5-L(x)-DOX conjugates ranged from -0.44 to -1.27, which is below the reported σ threshold (-0.37) required for binding to azoreductase enzymes. Results show that incubation of G5-L1-DOX (σ = -0.44), G5-L2-DOX (σ = -0.71), G5-L3-DOX (σ = -1.00), and G5-L4-DOX (σ = -1.27) conjugates with human liver microsomal (HLM) enzymes and the S9 fraction isolated from HepG2 hepatic cancer cells results in release of 4%-8%, 17%, 60%, and 100% of the conjugated DOX molecules, respectively. These results show that increasing the electronegativity (i.e. lower σ value) of L1-L4 azo-linkers increases their susceptibility to cleavage by azoreductase enzymes. Intracellular cleavage of G5-L(x)-DOX nanoconjugates, release of conjugated DOX molecules, and cytotoxicity correlated with conjugate's electronegativity (σ value) was investigated, with G5-L4-DOX conjugate exhibiting the highest toxicity towards hepatic cancer cells with an IC50 of 13 nm ± 5 nm in HepG2 cells. Cleavage of G5-L(x)-DOX conjugates was specific to hepatic cancer cells as shown by low non-specific DOX release upon incubation with non-enzymatic insect proteins and the S9 fraction isolated from rat cardiomyocytes. These enzyme-activated G5-L(x)-DOX conjugates represent a drug delivery platform that can achieve tunable and cell-specific release of the loaded cargo in hepatic cancer cells.

Modification of polydivinylbenzene microspheres by a hydrobromination/click-chemistry protocol and their protein-adsorption properties.

Hydrophobic- and/or hydrophilic-polymer-grafted PDVB microspheres are synthesized by the combination of hydrobromination and click-chemistry processes. The modified-PDVB microspheres and the intermediates at various stages of synthesis are characterized using GPC, ¹H NMR and FTIR spectroscopy and TGA analysis. Use of the microspheres as a support matrix for reversible protein immobilization via adsorption is investigated. The system parameters such as the adsorption conditions (i.e., enzyme concentration, medium pH) and desorption are studied and evaluated with regards to the biocatalytic activity and adsorption capacity.

Phenacyl onium salt photoinitiators: synthesis, photolysis, and applications.

Onium salts, namely sulfonium, phosphonium, ammonium, and pyridinium salts containing phenacyl group are photoinitiators appropriate for the polymerization of monomers such as oxiranes and vinyl ethers, which are not polymerizable by a free-radical mechanism. The initiation is accomplished by direct or indirect (sensitized) photolysis of the salts. Depending on the type of the salt, the direct photoinitiation of cationic polymerization involves reversible or irreversible processes. The photolysis of phenacylsulfonium compounds proceeds by a reversible process, while the other types undergo irreversible photolysis leading to complete fragmentation of the photoinitiator. An additionally useful tool, namely photosensitized generation of initiating species enlarges the versatility of these salts as photoinitiators. Photoinitiated free-radical and zwitterionic polymerizations by using phenacyl-type salts are also addressed. Keto-enol tautomerization of phenacyl pyridinium salts is discussed. Moreover, an interesting application concerning in situ synthesis of clay-poly(methyl methacrylate) nanocomposites with the aid of the phenacyl anilinium salt-based photopolymerization technique is noted.