Development of Acoustically Active Nanocones Using the Host–Guest Interaction as a New Histotripsy Agent

WOS: 000460237300188Histotripsy is a noninvasive and nonthermal ultrasound ablation technique, which mechanically ablates the tissues using very short, focused, high-pressured ultrasound pulses to generate dense cavitating bubble cloud. Histotripsy requires large negative pressures (>= 28 MPa) to generate cavitation in the target tissue, guided by real-time ultrasound imaging guidance. The high cavitation threshold and reliance on real-time image guidance are potential limitations of histotripsy, particularly for the treatment of multifocal or metastatic cancers. To address these potential limitations, we have recently developed nanoparticle-mediated histotripsy (NMH) where perfluorocarbon (PFC)-filled nanodroplets (NDs) with the size of similar to 200 nm were used as cavitation nuclei for histotripsy, as they are able to significantly lower the cavitation threshold. However, although NDs were shown to be an effective histotripsy agent, they pose several issues. Their generation requires multistep synthesis, they lack long-term stability, and determination of PFC concentration in the treatment dose is not possible. In this study, PFC-filled nanocones (NCs) were developed as a new generation of histotripsy agents to address the mentioned limitations of NDs. The developed NCs represent an inclusion complex of methylated beta-cyclodextrin as a water-soluble analog of beta-cyclodextrin and perfluorohexane (PFH) as more effective PFC derivatives for histotripsy. Results showed that NCs are easy to produce, biocompatible, have a size <50 nm, and have a quantitative complexation that allows us to directly calculate the PFH amount in the used NC dose. Results further demonstrated that NCs embedded into tissue-mimicking phantoms generated histotripsy cavitation "bubble clouds" at a significantly lower transducer amplitude compared to control phantoms, demonstrating the ability of NCs to function as effective histotripsy agents for NMH.LOreal-UNESCO "For Women in Science" ProgramThe author (Y.Y.D.) thanks LOreal-UNESCO "For Women in Science" Program for financial support of this work

Development of Nanodroplets for Histotripsy-Mediated Cell Ablation

This report describes the synthesis of amphiphilic copolymers (ABC-1 and ABC-2) composed of a hydrophilic poly­(ethylene glycol) (PEG) block, a central poly­(acrylic acid) (PAA) block, and a random copolymer of heptadecafluorodecyl methacrylate (HDFMA) and methyl methacrylate (MMA) forming the hydrophobic block, which are used to form nanodroplets for ultrasound-mediated cell ablation. Specifically, the effect of molecular weight of PEG and P­(HDFMA-<i>co</i>-MMA) blocks on polymer’s ability to self-assemble around a variable amount (0%, 1%, and 2% v/v) of perfluoropentane (PFP) forming nanodroplets is investigated. The ability of different nanodroplets formulations embedded with a monolayer of red blood cells (RBCs) in tissue-mimicking agarose phantoms to initiate and sustain a bubble cloud in response to ultrasound treatments with different acoustic pressures and the associated ablation of RBCs were also investigated. Results show that ABC-1 polymer composed of a 2 kDa PEG block and a 6.7 kDa P­(HDFMA-<i>co</i>-MMA) block better encapsulate the PFP core compared to ABC-2 polymer composed of a 5 kDa PEG block and 11.4 kDa P­(HDFMA-<i>co</i>-MMA) block. Further, the ablative capacity indicated by the damage area in the RBCs monolayer increased with the increase in PFP content and reached its maximum with the nanodroplets formulated using ABC-1 polymer and encapsulating 2% v/v PFP. The nanodroplets formulated using ABC-1 polymer and loaded with 2% PFP produced the cavitation cloud and exhibited their ablative effect at an acoustic pressure that is 2.5-fold lower than the acoustic pressure needed to generate the same effect using a histotripsy (ultrasound) pulse alone, which indicates the ability of these nanodroplets to achieve targeted and self-limiting fractionation of disease cells while sparing neighboring healthy ones. Results also show that effective nanodroplets maintained their size and concentration upon incubation with bovine serum albumin at 37 °C for 24 h, which indicates their stability in physiologic conditions and their promise for in vivo cancer cell ablation

EFFECTS OF ULTRASOUND FREQUENCY ON NANODROPLET-MEDIATED HISTOTRIPSY

Nanodroplet-mediated histotripsy (NMH) is a targeted ultrasound ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells for targeted tumor ablation. In a previous study, it was reported that by use of extremely short, high-pressure pulses, histotripsy cavitation bubbles were generated in regions containing nanodroplets at significantly lower pressure (similar to 10.8 MPa) than without nanodroplets (similar to 28 MPa) at 500 kHz. Furthermore, it was hypothesized that lower frequency would improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion, and decreasing the cavitation threshold. In this study, we investigated the effects of ultrasound frequency (345 kHz, 500 kHz, 1.5 MHz, and 3 MHz) on NMH. First, the NMH cavitation threshold was measured in tissue phantoms with and without nanodroplets, with results indicating that the NMH threshold was significantly below the histotripsy intrinsic threshold at all frequencies. Results also indicated that the NMH threshold decreased at lower frequency, ranging from 7.4 MPa at 345 kHz to 13.2 MPa at 3 MHz. In the second part of this study, the effects of frequency on NMH bubble expansion were investigated, with results indicating larger expansion at lower frequency, even at a lower pressure. In the final part of this study, the ability of perfluoropentane-encapsulated nanodroplets to act as sustainable cavitation nuclei over multiple pulses was investigated, with results indicating that the nanodroplets are destroyed by the cavitation process and only function as cavitation nuclei for the first few pulses, with this effect being most pronounced at higher frequencies. Overall, the results of this study support our hypothesis that using a lower frequency will improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion and decreasing the cavitation threshold. (E-mail: [email protected]) (C) 2015 World Federation for Ultrasound in Medicine & Biology

Modification of Polydivinylbenzene Microspheres by a Hydrobromination/Click-Chemistry Protocol and their Protein-Adsorption Properties

WOS: 000285932600016PubMed ID: 20945305Hydrophobic-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, H-1 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.Istanbul Technical University (ITU)Istanbul Technical University; Ege UniversityEge UniversityThis work was supported by Istanbul Technical University (ITU) and Ege University research funds

Synergistic Combination of Small Molecule Inhibitor and RNA Interference against Antiapoptotic Bcl‑2 Protein in Head and Neck Cancer Cells

B-cell lymphoma 2 (Bcl-2) is an antiapoptotic protein that is overexpressed in head and neck squamous cell carcinomas, which has been implicated in development of radio- and chemoresistance. Small molecule inhibitors such as AT-101 (a BH3-mimetic drug) have been developed to inhibit the antiapoptotic activity of Bcl-2 proteins, which proved effective in restoring radio- and chemo-sensitivity in head and neck cancer cells. However, high doses of AT-101 are associated with gastrointestinal, hepatic, and fertility side effects, which prompted the search for other Bcl-2 inhibitors. Short interfering RNA (siRNA) proved to inhibit antiapoptotic Bcl-2 protein expression and trigger cancer cell death. However, transforming siRNA molecules into a viable therapy remains a challenge due to the lack of efficient and biocompatible carriers. We report the development of degradable star-shaped polymers that proved to condense anti-Bcl-2 siRNA into “smart” pH-sensitive and membrane-destabilizing particles that shuttle their cargo past the endosomal membrane and into the cytoplasm of head and neck cancer cells. Results show that “smart” anti-Bcl-2 particles reduced the mRNA and protein levels of antiapoptotic Bcl-2 protein in UM-SCC-17B cancer cells by 50–60% and 65–75%, respectively. Results also show that combining “smart” anti-Bcl-2 particles with the IC₂₅ of AT-101 (inhibitory concentration responsible for killing 25% of the cells) synergistically inhibits cancer cell proliferation and increases cell apoptosis, which reduce the survival of UM-SCC-17B cancer cells compared to treatment with AT-101 alone. Results indicate the therapeutic benefit of combining siRNA-mediated knockdown of antiapoptotic Bcl-2 protein expression with low doses of AT-101 for inhibiting the growth of head and neck cancer cells

Formulation of Acid-Sensitive Micelles for Delivery of Cabazitaxel into Prostate Cancer Cells

We report the synthesis of an amphiphilic triblock copolymer composed of a hydrophilic poly­(ethylene glycol) (PEG) block, a central poly­(acrylic acid) (PAA) block, and a hydrophobic poly­(methyl methacrylate) (PMMA) block using atom transfer radical polymerization technique. We examined the self-assembly of PEG-<i>b</i>-PAA-<i>b</i>-PMMA copolymers in aqueous solutions forming nanosized micelles and their ability to encapsulate hydrophobic guest molecules such as Nile Red (NR) dye and cabazitaxel (CTX, an anticancer drug). We used 2,2β′-(propane-2,2-diylbis­(oxy))-diethanamine to react with the carboxylic acid groups of the central PAA block forming acid-labile, shell cross-linked micelles (SCLM). We investigated the loading efficiency and release of different guest molecules from non-cross-linked micelles (NSCLM) and shell cross-linked micelles (SCLM) prepared by reacting 50% (SCLM-50) and 100% (SCLM-100) of the carboxylic acid groups in the PAA in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions as a function of time. We examined the uptake of NR-loaded NSCLM, SCLM-50, and SCLM-100 micelles into PC-3 and C4-2B prostate cancer cells and the effect of different micelle compositions on membrane fluidity of both cell lines. We also investigated the effect of CTX-loaded NSCLM, SCLM-50, and SCLM-100 micelles on the viability of PC-3 and C4-2B cancer cells compared to free CTX as a function of drug concentration. Results show that PEG-<i>b</i>-PAA-<i>b</i>-PMMA polymers form micelles at concentrations ≥11 μg/mL with an average size of 40–50 nm. CTX was encapsulated in PEG-<i>b</i>-PAA-<i>b</i>-PMMA micelles with 55% loading efficiency in NSCLM. <i>In vitro</i> release studies showed that 30% and 85% of the loaded CTX was released from SCLM-50 micelles in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions over 30 h, confirming micelles’ sensitivity to solution pH. Results show uptake of NSCLM and SCLM into prostate cancer cells delivering their chemotherapeutic cargo, which triggered efficient cancer cell death. PEG-<i>b</i>-PAA-<i>b</i>-PMMA micelles were not hemolytic and did not cause platelet aggregation, which indicate their biocompatibility

Noninvasive Ablation of Prostate Cancer Spheroids Using Acoustically-Activated Nanodroplets

We have developed acoustically activated nanodroplets (NDs) using an amphiphilic triblock copolymer, which self-assembles and encapsulates different perfluorocarbons including perfluoropentane (PFP) and perfluorohexane (PFH). Applying histotripsy pulses (i.e., short, high pressure, ultrasound pulses) to solutions of PFP- and PFH-NDs generated bubble clouds at a significantly reduced acoustic pressure compared to the cavitation pressure observed for histotripsy treatment alone. In this report, we summarize the results of combining histotripsy at low frequency (345 and 500 kHz) with PFP-NDs and PFH-NDs on the ablation of PC-3 and C4-2B prostate cancer cells. Using custom built histotripsy transducers coupled to a microscope and a high speed recording camera, we imaged the generation of a cavitation bubble cloud in response to different ultrasound regimes in solution and in tissue-mimicking gel phantoms. We quantified the associated ablation of individual cancer cells and 3D spheroids suspended in solution and embedded in tissue phantoms to compare the ablative capacity of PFP-NDs and PFH-NDs. Results show that histotripsy pulses at high acoustic pressure (26.2 MPa) ablated 80% of prostate cancer spheroids embedded in tissue-mimicking gel phantoms. In comparison, combining histotripsy pulses at a dramatically lower acoustic pressure (12.8 MPa) with PFP-NDs and PFH-NDs caused an ablation of 40% and 80% of the tumor spheroid volumes, respectively. These results show the potential of acoustically activated NDs as an image-guided ablative therapy for solid tumors and highlight the higher ablative capacity of PFH-NDs, which correlates with the boiling point of the encapsulated PFH and the stability of the formed bubble cloud

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, whenNMHtherapy 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 similar to 29 degrees C, surface tension similar to 9.5 mN/m) and perfluorohexane (PFH, boiling point similar to 56 degrees C, surface tension similar to 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 (similar to 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. (E-mail: [email protected] or http://www.bme.umich.edu/centlab.php) (C) 2016World Federation for Ultrasound in Medicine & Biology

Formulation of acid-sensitive micelles for delivery of cabazitaxel into prostate cancer cells

WOS: 000373550600023PubMed ID: 26977718We report the synthesis of an amphiphilic triblock copolymer composed of a hydrophilic polyethylene glycol) (PEG) block, a central poly(acrylic acid) (PAA) block, and a hydrophobic poly(methyl methacrylate) (PMMA) block using atom transfer radical polymerization technique. We examined the self-assembly of PEG-b-PAA-b-PMMA copolymers in aqueous solutions forming nanosized micelles and their ability to encapsulate hydrophobic guest molecules such as Nile Red (NR) dye and cabazitaxel (CTX, an anticancer drug). We used 2,2 beta'-(propane-2,2-diylbis(oxy))-diethanamine to react with the carboxylic acid groups of the central PAA block forming acid-labile, shell cross-linked micelles (SCLM). We investigated the loading efficiency and release of different guest molecules from non-cross-linked micelles (NSCLM) and shell cross-linked micelles (SCLM) prepared by reacting 50% (SCLM-50) and 100% (SCLM-100) of the carboxylic acid groups in the PAA in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions as a function of time. We examined the uptake of NR-loaded NSCLM, SCLM-50, and SCLM-100 micelles into PC-3 and C4-2B prostate cancer cells and the effect of different micelle compositions on membrane fluidity of both cell lines. We also investigated the effect of CTX-loaded NSCLM, SCLM-50, and SCLM-100 micelles on the viability of PC-3 and C4-2B cancer cells compared to free CTX as a function of drug concentration. Results show that PEG-b-PAA-b-PMMA polymers form micelles at concentrations >= 11 mu g/mL with an average size of 40-50 nm. CTX was encapsulated in PEG-b-PAA-b-PMMA micelles with 55% loading efficiency in NSCLM. In vitro release studies showed that 30% and 85% of the loaded CTX was released from SCLM-50 micelles in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions over 30 h, confirming micelles' sensitivity to solution pH. Results show uptake of NSCLM and SCLM into prostate cancer cells delivering their chemotherapeutic cargo, which triggered efficient cancer cell death. PEG-b-PAA-b-PMMA micelles were not hemolytic and did not cause platelet aggregation, which indicate their biocompatibility.National Ministry of Education of the Republic of Turkey [1416]; Ministry of Higher Education of the Arab Republic of EgyptO.A. wishes to recognize the financial support of the National Ministry of Education of the Republic of Turkey (Program #:1416). I.Y. recognizes the financial support of the Ministry of Higher Education of the Arab Republic of Egypt through the Joint Supervision Fellowship Program. I.Y. also recognizes the support of Dr. Ahmed Fadda, Dr. ElSayed Afsah, and Dr. Ibrahim El-Sherbiny for serving as the dissertation committee at Mansoura University, Mansoura, Egypt. The authors thank Dr. Daniel Eitzman and Dr. Jinsang Kim at the University of Michigan for granting access to the Aggro-Link Data Reduction System (Chrono-log Corporation, Havertown, PA) and the QM4 Fluorescence Spectrophotometer (PerkinElmer, Waltham, MA) used to measure platelets aggregation and changes in albumin fluorescence, respectively. The authors also thank Mr. Ahmet Emrehan Emre for his technical assistance with electron microscopy imaging