Noninvasive Ablation of Prostate Cancer Spheroids Using Acoustically

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Non-invasive Ablation of Prostate Cancer Spheroids Using Acoustically-Activated Nano-Droplets Omer Aydin, Eli Vlaisavljevich, Yasemin Yuksel Durmaz, Zhen Xu, and Mohamed E.H. ElSayed Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00617 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Molecular Pharmaceutics

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Non-invasive Ablation of Prostate Cancer Spheroids Using Acoustically-Activated Nano-

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Droplets

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Omer Aydin1, Eli Vlaisavljevich1, Yasemin Yuksel Durmaz1,2, Zhen Xu1,3*, and Mohamed E.H.

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ElSayed1,4*

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1 - University of Michigan, Department of Biomedical Engineering, Ann Arbor, MI 48109, USA

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2 - Istanbul Medipol University, School of Engineering and Natural Sciences, Department of

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Biomedical Engineering, Beykoz, Istanbul, 34810, Turkey

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3 - University of Michigan, Department of Pediatrics and Communicable Diseases, Division of

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Pediatric Cardiology, Ann Arbor, MI 48109, USA

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4 - University of Michigan, Macromolecular Science and Engineering Program, Ann Arbor, MI

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48109, USA

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*Corresponding Author:

*Corresponding Author:

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Zhen Xu, Ph.D.

Mohamed E.H. ElSayed, Ph.D.

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University of Michigan

University of Michigan

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Department of Biomedical Engineering

Department of Biomedical Engineering

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2107 Carl A. Gerstacker Building

1101 Beal Ave.

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2200 Bonisteel Boulevard

2150 Lurie Biomedical Engineering Bldg

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Ann Arbor, MI 48109, USA

Ann Arbor, MI 48109, USA

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Phone: + 1 (734) 647-4961

Phone: + 1 (734) 615-9404

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Fax: + 1 (734) 939-1905

Fax: + 1 (734) 647-4834

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E-mail: [email protected]

E-mail: [email protected]

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Web:www.bme.umich.edu/labs/xulab/

Web: www.bme.umich.edu/centlab.php

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ACS Paragon Plus Environment


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Abstract

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We have developed acoustically-activated nanodroplets (NDs) using an amphiphilic triblock

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copolymer, which self-assembles and encapsulates different perfluorocarbon including

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perfluoropentane (PFP) and perfluorohexane (PFH). Applying histotripsy pulses (i.e. short, high

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pressure, ultrasound pulses) to solutions of PFP- and PFH-NDs generated bubbles clouds at a

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significantly reduced acoustic pressure compared to the cavitation pressure observed for

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histotripsy treatment alone. In this report, we summarize the results of combining histotripsy at

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low frequency (345 and 500 kHz) with PFP-NDs and PFH-NDs on the ablation of PC-3 and C4-

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2B prostate cancer cells. Using custom built histotripsy transducers coupled to a microscope and

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a high speed recording camera, we imaged the generation of a cavitation bubble cloud in

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response to different ultrasound regimes in solution and in tissue-mimicking gel phantoms. We

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quantified the associated ablation of individual cancer cells and 3D spheroids suspended in

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solution and embedded in tissue phantoms to compare the ablative capacity of PFP-NDs and

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PFH-NDs. Results show that histotripsy pulses at high acoustic pressure (26.2 MPa) ablated 80%

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of prostate cancer spheroids embedded in tissue-mimicking gel phantoms. In comparison,

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combining histotripsy pulses at a dramatically lower acoustic pressure (12.8 MPa) with PFP-NDs

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and PFH-NDs caused an ablation of 40% and 80% of the tumor spheroid volumes, respectively.

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These results show the potential of acoustically-activated NDs as an image-guided ablative

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therapy for solid tumors and highlight the higher ablative capacity of PFH-NDs, which correlates

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with the boiling point of the encapsulated PFH and the stability of the formed bubble cloud.

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Keywords:

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Perfluorocarbon, Targeted cell ablation

Acoustically-activated

nanodroplets,

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Histotripsy,


Cavitation

bubble

cloud,

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INTRODUCTION

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The widespread use of prostate-specific antigen (PSA) screening and the increase in life

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expectancy have increased the number of men diagnosed with prostate cancer 1. These patients

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are routinely managed with definitive therapy including radical prostatectomy (RP) or radiation

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therapy (RT), which are associated with significant complications and side effects such as

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incontinence and erectile dysfunction that greatly deteriorate patient’s quality of life 2, 3. Analysis

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of RP specimen reveals that 49% of men undergoing RP show insignificant or indolent cancer

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(i.e. organ-confined cancer lesion < 0.5 mL) 4 compared to 73% of the patients initially enrolled

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in active surveillance who undergo RP and reveal a significant cancer on RP specimens 5.

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Analysis of RP specimens showed that 38% were unifocal 6 and 80% of the total tumor volume

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in multifocal disease arose from the index tumor with extracapsular extension present in only

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28% of these patients 7. These results clearly indicate that a large population of prostate cancer

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patients have a biologically unifocal disease, which suggests the potential for using focal therapy

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(i.e. targeted ablation of the cancer lesions) as an alternative treatment strategy to RP 8. Success

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of focal therapy depends on the ability to detect and image tumor tissue, target the cancer lesions,

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monitor the therapeutic response, and assess the results 8.

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Histotripsy is a noninvasive tissue ablation technique that controllably fractionates soft tissue

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using a dense cavitation bubble cloud generated by short duration ultrasound pulses under high

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acoustic pressure (~25-30 MPa)

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and maintain a dense cavitation bubble cloud over multiple pulses (often >100) until the treated

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tissue is completely fractionated into a liquid homogenate with complete loss of the cellular

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structure

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be the rapid expansion and collapse of cavitation bubbles, which fractionates soft tissues into an

10-12

9-11

. Earlier research shows that histotripsy treatments initiate

. The process by which histotripsy achieves tissue fractionation has been shown to

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acellular homogenate by inducing very large strains to the adjacent tissue structures 13. Currently,

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non-invasive tissue removal using histotripsy is being clinically evaluated in benign prostatic

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hyperplasia

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the promise of histotripsy as an ablation therapy, it remains a non-targeted approach that requires

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high acoustic pressure, which hinders its use in patients with deeply located tumor nodules or

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micro-metastases.

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, deep vein thrombosis

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, congenital heart disease

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, and cancer

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. Despite

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To address the limitations of histotripsy, our group has developed acoustically-activated

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nanodroplets to achieve selective mechanical fractionation of diseased tissue at low acoustic

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pressure. Specifically, we designed and synthesized an amphiphilic triblock copolymer

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composed of a hydrophilic poly(ethylene glycol) (PEG) block, a central poly(acrylic acid) (PAA)

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block, and a

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(P(HDFMA-co-MMA)) block, which proved to self-assemble and encapsulate 2% v/v

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perfluorocarbon (PFC) forming acoustically-activated nanodroplets

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cross-linker to covalently “stitch” the central PAA blocks forming a flexible polymer shell that

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displays a brush of biocompatible PEG chains, which can be further functionalized to display

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different ligands (e.g. sugars, peptides, antibodies, or aptamers) to allow selective targeting to

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specific disease sites. Our earlier results show that the nanodroplets rapidly expand forming

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microbubbles (>500µm) before energetically collapsing at an acoustic pressure that is 2.5-fold

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lower than the acoustic pressure needed to generate the same bubble cloud using the histotripsy

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pulse alone

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microbubbles and their subsequent cavitation using therapeutic ultrasound, which is the basis for

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their potential use in ultrasound-guided ablation therapy

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changing the PFC core from perfluoropentane (PFP, boiling point ~29 ºC, surface tension ~9.5

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hydrophobic poly(heptadecafluorodecyl methacrylate-co-methyl methacrylate)

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. We used a chemical

. Results also confirm the feasibility of imaging nanodroplets expansion into

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18, 20, 21


. We investigated the effect of

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mN/m) to perfluorohexane (PFH, boiling point ~56 ºC, surface tension ~11.9 mN/m) on the

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acoustic and ablative properties of PFP-loaded and PFH-loaded nanodroplets

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loaded and PFH-loaded nanodroplets exhibited a significantly reduced cavitation threshold

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compared to the cavitation threshold of histotripsy alone. However, the PFH-loaded nanodroplets

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maintained the bubble cloud over 1000 pulses compared to PFP-loaded nanodroplets, which

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were destroyed by the cavitation process in less than 50 pulses likely due to the lower boiling

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point of the PFP core 20, 22. Further, the PFH-loaded nanodroplets generated well-defined ablation

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lesions in the RBCs layers sandwiched in tissue-mimicking gel phantoms indicating a stronger

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ablative capacity compared to PFP-loaded nanodroplets 23.

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. Both PFP-

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In this study, we expand on our previous research studying nanodroplet-mediated histotripsy

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(NMH) and investigate the feasibility of achieving NMH ablation of PC-3 and C4-2B prostate

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cancer cells in vitro using PFP- and PFH-loaded nanodroplets at acoustic pressures significantly

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below the regular histotripsy. When using a single ultrasound pulse of 99.9 % St. Luis, MO), resazurin sodium salt

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(Sigma-Aldrich, St. Luis, MO), PEG Bioultra 35,000 (Sigma-Aldrich, St. Luis, MO), Dextran

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500,000 (Spectrum, Gardena, Ca), Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Luis, MO),

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Ultrapure Agarose (Invitrogen, Carlsbad, Ca) were used as delivered. RPMI medium 1640, fetal

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bovine serum (FBS), 0.25% trypsin/0.20% ethylene diamine teraacetic acid (EDTA), phosphate

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buffered saline (PBS), penicillin/streptomycin/ amphotericin, sodium pyruvate, nonessential

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amino acid solutions, the Live/Dead cytotoxicity Kit for mammalian cells (Calcein AM and

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Ethidium homodimer-1), and agarose were purchased from Invitrogen Corporation (Carlsbad,

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CA). Perfluoropentane (PFP, Alfa Aesar, 97% ca. 85% n-isomer) and perfluorohexane (PFH,

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SynQuest Lab, > 98%) were directly used without prufication. N-hydroxy succinimide (NHS, 97

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%), and N-(3-Dimethylaminopropyl)-N ethylcarbodiimide hydrochloride (EDC, >98 %) were

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purchased from Fluka (St. Luis, MO). T-75 flasks, Costar 96-well plates, and cell culture

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supplies were purchased from Corning Inc. (Corning, NY).

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Instruments A 500 MHz Varian Mercury system (Palo Alto, CA) was used to record all the 1H NMR and

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13

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(GPC) analysis was done using a Viscotek GPCmax Autosampler system consisting of a pump

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and Water 2414 refractive index (RI) detector. The molecular weight and molecular weight

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distribution of synthesized polymers were determined based on their elution volume on a

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Styragel HR 4E column compared to a series of poly(methyl methacrylate) standards

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(PolyAnalitik Inc, Canada) using THF as a mobile phase at a flow rate of 1 mL/min at 35 oC.

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The data was analyzed using Viscotek OmniSEC Omni-01 software. Perkin-Elmer FT-IR

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Spectrum 4100 type A machine was used to confirm the azidation of different polymer blocks.

C NMR spectra of polymer blocks at room temperature. Gel permeation chromatography

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Formulation of PFP- and PFH-Loaded Nanodroplets

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The amphiphilic, triblock PEG45-b-PAA12-b-P(HDFMA8-co-MMA20) copolymer was used to

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prepare PFP- and PFH-loaded nanodroplets with equal PFC loading (2% v/v) following

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published protocols

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synthesized using a combination of atom transfer radical polymerization (ATRP) and “click”

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coupling techniques followed by purification and confirmation of polymer composition using

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GPC, 1H NMR, and MALDI techniques following established protocols

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different nanodroplets started by dissolving the copolymer in tetrahydrofuran (THF) (0.2% w/v)

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and cooling down the polymer solution to 0 °C before adding the PFP (2% v/v) or PFH (2% v/v)

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. Briefly, the PEG45-b-PAA12-b-P(HDFMA8-co-MMA20) polymer was

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18-20

. Formulation of


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while vigorously stirring the copolymer-perfluorocarbon mixture (Figure 1). An equal volume of

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deionized water was added drop-wise to the polymer/PFC mixture to initiate micelle formation

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while keeping the solution in an ice bath for 1h before transferring the micelles solution to a

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dialysis bag (MWCO of 1 KDa) and dialyzing overnight against ice-cold 2-(N-

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morpholino)ethanesulfonic acid monohydrate solution (MES buffer, pH 5.5) to remove the THF

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and obtain

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formulation was transferred to a round bottom flask and mixed with 2,2`-(ethylenedioxy)-

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bis(ethylamine) to cross-link the carboxylic acid groups in the central PAA block in the polymer

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backbone via NHS/EDC coupling chemistry forming the cross-linked nanodroplets with a

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flexible polymer shell. Shell cross-linked nanodroplets were dialyzed against ice-cold water for

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12 hours to remove the unreacted cross-linker and reaction byproducts.

a milky solution of uncross-linked PFP- and PFH-loaded nanodroplets. Each

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Nanodroplets Characterization

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Size and concentration of PFP- and PFH-loaded nanodroplets were measured using the

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Nanoparticle Tracking Analysis (NTA) at 37°C. The NanoSight™ LM10 system (Malvern

16

Instruments, Amesbury, UK) equipped with a temperature-controlled 405 nm laser module, a

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high sensitivity scientific Complementary Metal Oxide Semiconductor (sCMOS) camera

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(Hamamatsu, Orca, Hamamatsu City, Japan), and a syringe pump was used for the collection of

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NTA data. Briefly, nanodroplets solutions were diluted to the appropriate concentration using

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deionized water before imaging where 60s videos (5 videos per sample) were acquired and

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analyzed using the NTA software (Version 3.0, Build 0066) at 37 °C. We plotted nanodroplets

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concentration as a function of droplet size with the error bars representing the standard deviation

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of the repeat measurements for each sample. The mean size and standard deviation values

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obtained by the NTA software correspond to arithmetic values calculated with the sizes of all

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particles analyzed by the software. The error bars displayed on the graphs were obtained by the

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standard deviation of a minimum of five different samples.

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We investigated the stability of PFH-loaded nanodroplets NDs when incubated bovine serum

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albumin (BSA) as a model serum protein for 24 hours at 37 ᵒC while stirring by monitoring the

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change in droplets size and concentration using NTA. This analysis was done by mixing 900 µL

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of PFH-nanodroplets with 100 µL of BSA (20 µg/mL) and imaging droplets solution following

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our previously published protocol

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two tailed Student’s t tests where p < 0.05 indicates statistically significant difference between

10

19

. All statistical evaluations were carried out with unpaired

groups.

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Cell Culture

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PC-3 and C4-2B prostate cancer cells were cultured following published protocols 22. Briefly,

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both cell lines were cultured in RPMI-1640 supplemented with 10% FBS and 1%

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antibiotic/antimycotic solution. PC-3 and C4-2B cells were incubated at 37 °C and 5% CO2

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while changing the culture medium every other day and passaging the cells using 0.05%

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trypsin/EDTA solution after reaching 80% confluence. The passage number for PC-3 and C4-2B

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cells used in the cytotoxicity and ablation studies was in the range between 25 and 40.

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We used the Aqueous Two-Phase System (ATPS) to prepare PC-3 and C4-2B spheroids 24

20

following published protocols

with minor modifications. Briefly, 5.0% (w/w) of PEG (35,000

21

Da) and 12.8% (w/w) of dextran (500,000 Da) stock solutions were prepared in the culture

22

medium and kept at 37°C. The surface of flat-bottom 24-well plates and cell holders were coated

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with 300 µL and 650 µL of a boiled agarose solution 2% (w/v) in double distilled water,

9



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respectively, to get a non-adherent surface. We allowed the agarose solution to cool down to

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room temperature and solidify in 10 min before washing twice with PBS and adding 500 µL of

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5.0% (w/w) PEG solution to each well. PC-3 and C4-2B cells were prepared at a concentration

4

of 700,000 cells/µL in 12.8% (w/w) aqueous dextran before adding 0.8 µL of this cell solution to

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the PEG solution in each well. Given that PEG and dextran solutions are immiscible, we allowed

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the cells to aggregate and grow at the interface for 24-48 hours to get uniform spherical

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spheroids. We measured the horizontal and vertical diameters of each spheroid using an inverted

8

microscope (Olympus IX51, Melville, NY) before adding more culture medium and incubating

9

for longer periods of time to increase the size of the spheroids. The spheroids were ready for use

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in 2 days of culture under these conditions We confirmed the viability of the cultured spheroids

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by incubating them with a solution of 3 µM Calcein AM and ethidium homodimer-1 for 2 hours

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before washing excess dye using PBS and examining the spheroids under an Olympus IX51

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fluorescent microscope (Melville, NY) coupled with 130 W U-HGLGPS fluorescence light

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source and a TL4 power supply unit. Fluorescent images were captured and analyzed using the

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Olympus image software analySIS® where green and red fluorescence indicated live and dead

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cells, respectively.

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Nanodroplets Cytotoxicity

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We evaluated the non-specific toxicity PFP- and PFH-loaded nanodroplets by incubating

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them with PC-3 and C4-2B cells. Briefly, PC-3 and C4-2B cells were seeded at a seeding density

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of 10 x 103 cells/well in 96-well plates and allowed to adhere for 24 h before adding the

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nanodroplets at concentrations between 0-100 µg/mL and incubating for 48 h. At the end of the

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incubation period, the culture medium was removed and the cells were incubated with 200 µL of

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the Resazurin dye at a dilution of 1/10 for 4 h before measuring solution fluorescence (λex= 570

2

nm, λem= 590 nm) using the Fluoroskan Ascent FL plate reader (Thermo Fisher Scientific,

3

Waltham, MA). Fluorescence values were normalized to that observed with PC-3 and C4-2B

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cells incubated in the regular culture medium (control group) to calculate the percentage of

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viable cells in response to different treatments. The nanodroplets concentration that resulted in a

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statistically significant reduction in cell viability was considered cytotoxic.

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Ablation of PC-3 and C4-2B Cells and Spheroids in Solution

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A 345 kHz ultrasound transducer was designed to generate a histotripsy bubble cloud and

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image the cavitation of PC-3 and C4-2B cells and spheroids using a clinical ultrasound imaging

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system (Philips HDI 5000 Ultrasound system, Bothell WA, USA). The 345 kHz transducer was

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designed to generate short, high-pressure, 1-2 cycle histotripsy pulses and had a geometric focus

13

of 150 mm, an aperture size of 272 mm, and an effective f-number of 0.55. Elements were driven

14

in phase with a custom high-voltage pulser that was developed in-house. The pulser was

15

connected to a field-programmable gate array (FPGA) board (Altera DE1 Terasic Technology,

16

Dover, DE) specifically programmed for histotripsy pulsing. This setup allowed the transducers

17

to output short pulses consisting of less than two cycles. A fiber optic probe hydrophone built in-

18

house

19

pressure levels (p- > 23 MPa), the acoustic output could not be directly measured due to

20

cavitation at the fiber tip. Therefore, these pressures were estimated by a summation of the

21

output focal p- values from individual transducer elements with these approximations assuming

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minimal nonlinear distortion of the waveform occurring within the focal region. In a previous

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study 26, this estimated p- was found to be accurate within 15% compared to direct focal pressure

25

was used to measure the acoustic output pressure of the transducers. At high negative

11



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measurements in water and in a medium (1,3 butanediol) with a higher cavitation threshold. The

2

transducer was placed inside a tank of degassed water at room temperature (22°C) with the

3

elements completely submerged. A computer-positioning system was built to support the tube

4

containing the treatment solution and to adjust the position of the tube with respect to the

5

transducer focus. The desired concentration (2 x 108 particles/mL) of PFP- or PFH-NDs was

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added to the solution of 200,000 individual PC-3 or C4-2B prostate cancer and, in a separate

7

study to three PC-3 or C4-2B spheroids in 2 mL of PBS in the treated tube. Individual PC-3/C4-

8

2B cells and spheroids were suspended in PBS before being added to the rubber tube, which was

9

placed in the focal zone of the 345 kHz array transducer placed inside a degassed water tank at

10

room temperature. The tube was positioned in the focal zone of the 345 kHz array transducer

11

using a PC-controlled console and scanned from the bottom to the top and vice versa for 5

12

minutes to ensure the exposure of the while volume of the solution to the ultrasound treatment

13

(Figure 2). At the end of the 5 min ablation treatment, the solutions of PC-3 and C4-2B

14

cells/spheroids were collected and mixed with the resazurin dye to quantify the fraction of viable

15

cells following the same protocol described earlier and used to assess NDs toxicity. The number

16

of viable PC-3 and C4-2B cells was normalized to that incubation in PBS without exposure to

17

NDs or ultrasound treatments to calculate the effect of different treatments on the viability of

18

individual cancer cells and 3D spheroids.

19 20

Visualizing the Ablation of PC-3 and C4-2B Spheroids in a Tissue Mimicking Environment

21

We investigated the effect of PFP- and PFH-NDs on PC-3 and C4-2B spheroids in agarose 27-29

22

phantoms as an accepted 3D model of cancer cell-tissue matrix interactions

23

phantoms containing 2% (w/v) agarose were prepared by slowly mixing agarose powder into

12


. Tissue

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PBS heated to the boiling temperature and keeping the solution stirring on a hot plate until the

2

gel turned completely transparent and allowed to boil for an additional 10 minutes. Agarose

3

solutions were allowed to cool down before being degassed under a partial vacuum (~20 kPa,

4

absolute) for 30 minutes. Agarose phantoms containing the nanodroplets were prepared by

5

slowly adding the desired volume of the nanodroplets solution into the cold agarose solution

6

while stirring before pouring into rectangular polycarbonate holders with acoustic windows and

7

keeping this mixture at room temperature to allow the agarose solution to solidify forming tissue

8

phantoms with embedded PFP- or PFH-loaded nanodroplets or without nanodroplets (control

9

phantoms).

10

A 500 kHz ultrasound transducer was designed to generate a histotripsy bubble cloud and

11

image the cavitation of PC-3 and C4-2B cells and spheroids. The 500 kHz transducer was

12

designed to generate short, high-pressure, 1-2 cycle histotripsy pulses and had a geometric focus

13

of 40 mm, an aperture size of 71 mm, and an effective f-number of 0.56. Elements were driven in

14

phase with a custom high-voltage pulser that was developed in-house. The pulser was connected

15

to a field-programmable gate array (FPGA) board (Altera DE1 Terasic Technology, Dover, DE)

16

specifically programmed for histotripsy pulsing. This setup allowed the transducers to output

17

short pulses consisting of less than two cycles. A fiber optic probe hydrophone built in-house 25

18

was used to measure the acoustic output pressure of the transducers.

19

A high-speed 1 megapixel CCD camera (Phantom V210, Vision Research) with a frame rate

20

of 2000 frame per second (fps) was aligned above an inverted microscope (Nikon Eclipse Ti,

21

10X magnification) (Figure 3) to visualize the interactions between the generated bubble cloud

22

and the cancer cells over multiple pulses. The camera was externally triggered from the FPGA

23

signal board to synchronize recorded images with the histotripsy pulse at specific delay intervals.

13



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1

An exposure time of 20 µs was used for each recorded frame and the camera was triggered to

2

record images 24 µs after the arrival of each pulse. The 3D spheroids were formulated following

3

the ATPS method described earlier. These spheroids were cultured on agarose gels for 3 days at

4

37 °C and 5% CO2 before treating the cells with 100 histotripsy pulses applied at a pulse

5

repetition frequency of 1 Hz. The camera recorded 20 images after each pulse using a 10X

6

magnification lens. In approximately 3 of these images, the bubble cloud was still present while

7

cell displacement and deformation was observed in the following images since these effects

8

lasted significantly longer than the bubble cloud 13.

9

To examine the ability of PC-3 cells to regrow after NMH, the homogenate of PC-3

10

spheroids incubated with PFH-NDs and treated with histotripsy were collected after treatment

11

and centrifuged at 1000 rpm for 5 min. The collected homogenate was mixed with 500 µL of

12

fresh RPMI 1640 medium, seeded in a 24-well plate, and incubated for 48 hours under the

13

regular culture conditions. Untreated (no PFH-NDs and no histotripsy pulses) PC-3 spheroids

14

were processed following the same protocol and used as a control group. After 48 hours, the

15

ability of the cultured homogenate to grow viable cells was evaluated using the resazurin

16

viability assay coupled with microscopic examination using an inverted microscope (Olympus

17

IX51, Melville, NY) with a 4X objective lens.

18 19

RESULTS AND DISCUSSION

20

Formulation and Characterization of PFP- and PFH-Loaded Nanodroplets

21

Our previous reports show that PEG45-b-(PAA)12-b-P(HDFMA8-co-MMA20) triblock

22

copolymer can efficiently encapsulate PFP and PFH forming nanodroplets (NDs) 18-22, 30 and we

23

followed the same formulation protocol to obtain PFP- and PFH-loaded NDs for these studies.

14


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1

Results of the NTA shows that PFP-and PFH-loaded NDs have a similar average size and size

2

distribution profiles (Figure 4A, Table 1). Specifically, the average size of PFP-loaded NDs was

3

between 100 nm and 450 nm with the size of major peak < 300 nm (Figure 4A) and an average

4

diameter of 177 ± 1.9 nm (Table 1). In comparison, PFH-loaded NDs exhibited a slightly larger

5

size distribution between 100 nm and 600 nm with 3 peaks in the range of 200-300 nm, 300-450

6

nm, and 450-600 nm (Figure 4A) and an average diameter of 233.9 ± 3.9 nm (Table 1). The

7

larger size of the PFH-loaded NDs compared to PFP-loaded ones can be a result of the difference

8

in miscibility between PFP and PFH in the THF/copolymer mixture. Briefly, our experimental

9

observations indicate that PFP homogenously disperses in the THF/copolymer mixture during

10

NDs formulation whereas, PFH tends to phase separate from the THF/copolymer mixture. We

11

broke down PFH phase separation by drop-wise addition of water to the THF/polymer mixture

12

and vigorous mixing, which triggered breakdown of the PFH phase and mixing with the aqueous

13

phase coupled with self-assembly of the polymer forming PFH-loaded NDs. The hydrophilic

14

PEG corona stabilized both PFP- and PFH-loaded NDs and inhibited their aggregation or

15

precipitation in solution at 4 ᵒC and 25 ᵒC. Given that >99% PFP- and PFH-loaded NDs have an

16

average diameter < 400 nm, they are in the size range to preferentially extravasate across tumor’s

17

leaky vasculature and accumulate in tumor tissue with low risk of inducing pulmonary embolism

18

31, 32

.

19

Serum albumin is the major soluble blood protein, which adsorbs to the surface of particles in

20

the systemic circulation and labels them for hepatic clearance 22, 33. We investigated the stability

21

of the PFH-NDs upon incubating with bovine serum albumin (BSA) at 37 ᵒC for 24 h by

22

monitoring the change in NDs size and concentration using NTA. Results show that the average

23

diameter of PFH-NDs before incubating at 37 ᵒC (0 h) is 251.9 ± 11.2 nm, which dropped to

15



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1

221.5 ± 8.7 nm after incubating the NDs at 37 ᵒC for 24 hours (Figure 4B). This drop in average

2

NDs size is a result of the loss of the fraction of NDs with an average size ~500 nm, which

3

resulted in ~40% of NDs concentration probably due to loss of poorly encapsulated PFH in large

4

NDs. Incubation of PFH-NDs with BSA at 37℃ for 24h similarly reduced average NDs size to

5

228.7 ± 10.3 nm and NDs concentration was similar in the presence and absence of BSA (Figure

6

4B). These results indicate that the PEG corona limits the interaction of PFH-NDs with BSA,

7

which increased NDs against complete dissolution and aggregation in the presence of BSA as a

8

model serum protein at 37 ᵒC. It is worth noting that the size of PFP-NDs did not change under

9

similar experimental conditions but the concentration decreased by 20% at 37℃ regardless of the

10

presence or absence of BSA 19. These results indicate that PFP-NDs and PFH-NDs will exhibit

11

minimal interaction with serum proteins in vivo.

12 13

Nanodroplets Cytotoxicity

14

To investigate the toxicity of the nanodroplet particles, we performed in vitro cytotoxicity

15

experiments using a resazurin viability assay, which is a very common dye to show cell viability,

16

proliferation and cytotoxicity with a simple, rapid, sensitive, and reliable way. Basically, it relies

17

on reduction of resazurin (blue, nonfluorescent form) to resorufin (pink, highly fluorescent) by

18

viable cells. The metabolized form of the dye enters the cytosol and is converted to the

19

fluorescent form by mitochondrial enzyme activity by accepting the electron from NADPH,

20

FADH, NADH, and cytochromes34. Both prostate cancer cell lines were treated with a variety of

21

concentrations between 0 to 100 µg/mL of PFP or PFH-loaded nanodroplets calculated initial

22

polymer weight that added to the formulation and the end volume of the dialysis step in ND

23

formulation for 24 h under regular cell culture conditions compared to negative controls of non-

16


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1

treated cells and positive controls of Triton-x 100 treated cells. It is important to highlight the

2

maximum concentration that we used is 100 times more than used in the tube cell ablation

3

particle concentration. Further, results show that incubation of PFP- and PFH-loaded NDs with

4

PC-3 and C4-2B cells did not affect their viability up to a concentration of 100 µg/mL except

5

only PFH-NDs at the highest particle concentration (Figure 5). Overall, this result points to

6

biocompatible character of the particles. As a result, higher concentration or volume usage of the

7

nanodroplets is not expected to be an issue in vivo because of the particles cytotoxicity profile.

8 9

Ablation of PC-3 and C4-2B Cells and Spheroids in solution

10

We investigate the effect of combining individual prostate cancer cells and spheroids with

11

PFP- and PFH-loaded NDs in solution in a rubber tube and exposing this mixture to histotripsy

12

pulses as shown in Figure 2. We hypothesized that applying histotripsy pulses with peak

13

negative pressures of ~10.7–12.8 MPa would activate the PFP- and PFH-NDs resulting in

14

generation of a bubble cloud that would energetically collapse and cause the mechanical ablation

15

of individual cancer cells and 3D tumor spheroids. In comparison, PC-3/C4-2B cells/spheroids

16

mixed with the NDs but were not treated with histotripsy pulses and PC-3/C4-2B cells/spheroids

17

not mixed with the NDs but treated with histotripsy pulses will not experience ablative forces

18

and will remain viable after the treatment. To test this hypothesis, we added an equal number (2

19

× 108 droplets/mL) of PFP- and PFH-loaded NDs to the designated tubes before applying

20

histotripsy pulses to the center of the tube at a pulse repetition frequency of 10 Hz and peak

21

negative pressure of 10.7 MPa. The tubes containing NDs showed a bubble cloud in the center of

22

the tube (Figure 2) which was monitored in real time using a clinical ultrasound imaging system

23

(Supplementary Videos 1 & 2). In contrast, no cavitation bubble clouds were observed in the

17



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1

solution without NDs. Quantifying the changes in cell viability in response to different

2

treatments using the resazurin dye show the ablative capacity associated with each treatment

3

(Figure 6). Results show that PC-3 and C4-2B cells (Figure 6A) and spheroids (Figure 6B) that

4

were not incubated with NDs or treated with ultrasound exhibited 100% viability at the end of

5

the treatment and were used a reference for normalization of other treatments. Similarly,

6

incubating PC-3 and C4-2B cells (Figure 5A) and spheroids (Figure 5B) with PFP- and PFH-

7

NDs without shining ultrasound did not affect cell viability. However, exposure to 1-2 cycle

8

histotripsy pulses (10.7 MPa) in absence of NDs caused less than ~20% reduction in the viability

9

of both PC-3 and C4-2B cells (Figure 6A). By combining PFP- and PFH-NDs and exposure to

10

1-2 cycle histotripsy (10.7 MPa), significant ablation of PC-3 and C4-2B cells and spheroids was

11

observed (Figure 6). Specifically, combining the NDs with ultrasound resulted in ablation of 40-

12

70% and 60-70% of suspended PC-3 and C4-2B cells, respectively (Figure 6A). Similarly,

13

combining the NDs with ultrasound resulted in ablation of 40%-60% of PC-3 and C4-2B

14

spheroids (Figure 6B). Upon added nanodroplets to cell/spheroid samples with applied 10.7

15

MPa ultrasound, we got further ablation therapeutic effects. For example, PFP-nanodroplets with

16

ultrasound resulted in the death of almost 80% and 60% of the individual PC-3 and C4-2B cells,

17

respectively (Figure 6A). PFH-nanodroplet results showed cell death rates of 40% and 75% for

18

the PC-3 and C4-2B cells, respectively. On the other hand, more than 50% and 40% of the PC-3

19

and C4-2B spheroids were destroyed with PFP-nanodroplets, respectively. Moreover, the

20

therapeutic activity of PFH-nanodroplets on PC-3 and C4-2B spheroids reached to 70% and 60%

21

cell ablation, respectively (Figure 6B). The nanodroplets containing PFP or PFH resulted in

22

higher cavitation so these offer higher therapeutic activity in response to the histotripsy treatment

23

based on the viability assay results. It is worth noting that these ablation rates of cells/spheroids

18


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1

are obtained from treating a single location in a certain time interval. The cell ablation rates are

2

expected to be improved when using targeted NDs. In addition, by scanning the ultrasound

3

therapy focus to cover the entire tumor volume, we expect to achieve complete ablation of the

4

entire targeted tumor volume.

5 6

Visualizing the Ablation of PC-3 and C4-2B Spheroids in a Tissue Mimicking Environment

7

We investigated the ability of PFP- and PFH-loaded NDs to ablate PC-3 and C4-2B

8

spheroids in an environment that mimics the mechanical properties that exist in vivo. We utilized

9

the ATPS to grow millimeter-sized spheroids in 2 days, which were placed on an agarose gel and

10

allowed to grow and establish cell-gel interactions. We applied ultrasound pulses at peak

11

negative pressures of 12.8 MPa and 26.2 MPa to the spheroids layer. The formation of a bubble

12

cloud and the associated cell fractionation was monitored using high speed optical imaging after

13

each pulse (Supplementary Video 3). Results show that histotripsy pulses (26.2 MPa) and the

14

bubble cloud generated by the NDs at low acoustic pressure (12.8 MPa) fractionated the

15

spheroids in the targeted region, forming an acellular homogenate (Figure 7A-C). Using these

16

images, we quantified the ablation area in response to different treatments (Figure 7D). Results

17

show that applying US at acoustic pressure below the histotripsy intrinsic threshold (12.8 MPa)

18

in tissue phantoms that were not loaded with either NDs did not cause significant ablation

19

(500µm) in response to histotripsy treatment only in the test group. Figure 3. (A) A custom built microscope transducer-tank with a three-axis positioning system was built to investigate the ability of nanodroplet-mediated histotripsy to fractionate tumor spheroids. (B) Prostate cancer spheroids were aligned at the transducer focus using a micropositioner, and histotripsy pulses were applied to the spheroids which were embedded inside an agarose gel to mimic a three-dimension tissue structure. Figure 4. (A) The concentration and size distribution of PFP (blue line) and PFH (red line)loaded nanodroplets (B) Stability test of PFH-NDs in the presence or absence of BSA for 24 h at 37 ᵒC, PFH-NDs at 0h (blue line), PFH-NDs at 24h (red line), and PFH-NDs at 24h with presence of BSA (green line). All measurements were calculated using Nanoparticles Tracking Analysis (NTA) and NTA 3.0 build 66 software. Results are the average of five independent experiments ± standard error of the mean (error bars not shown for B to clearly seen the lines). Figure 5. Evaluation of in vitro toxicity of PFP- and PFH-loaded nanodroplets towards PC-3 (A) and C4-2B (B) prostate cancer cells as a function of concentration of the polymeric carrier (0100 µg/mL) with Triton-X 100 positive control. Results are the average of two independent

29



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Page 30 of 43

experiments with 5 replicates ± standard error of the mean. We compared cell viability under each condition to the viability of untreated cells (negative control) using one-way ANOVA with Tukey test where # for PFH denotes p ≤ 0.05, and ## denotes p ≤ 0.01. Figure 6. Effect of PFP and PFH-loaded nanodroplets on the viability of PC-3 and C4-2B cultured as individual cells (A) or 3D spheroids (B) upon exposure to 1-2 cycle histotripsy pulses (10.7 MPa) applied with a pulse repetition frequency of 345 kHz. Cell viability was measured using Resazurin viability assay and results were normalized to untreated cells (negative control; – NDs and – US). Results are the average of four experiments ± standard error of the mean. We compared cell viability under each condition to the viability of untreated cells (negative control) using one-way ANOVA with Tukey test where * denotes p ≤ 0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, and **** denotes p ≤ 0.0005. Figure 7. 3D tumor model cell-bubble results demonstrate nanodroplet-mediated histotripsy can fractionate prostate cancer cells into accellular debris. A PC-3 prostate cancer spheroid was cultured on an agarose tissue phantom to mimic cells inside a tissue extracellular matrix. 100 histotripsy pulses were applied to the call layer containing nanodroplets at a pressure significantly below the histotripsy threshold without nanodroplets. (A) Digitized video capture images of NMH during treatment, cavitation bubbles were observed at the transducer focus and cells were observed to be mechanically ruptured. After treatment, no/less cells remained intact in the region in which the histotripsy bubble cloud was observed. (B) PC-3 3D spheroid NMH, the first column is the treatment of no ND with 12.8 MPa of US pressure, the second column is the PFP NDs mediated histotripsy with 12.8 MPa of US pressure, the third column is no ND with 26.2 MPa of US pressure, and the forth column is the PFH NDs mediated histotripsy with 12.8 MPa of US pressure. Each row is separated with before, during, and after US treatments. The

30


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spheroid border, cavitation bubbles, and ablated spheroid section were encircled with red, white, and yellow colors, respectively to be easily distinguished the treatment efficiency. The cross bar is 200 µm. (C) C4-2B 3D spheroid NMH, the first column is the treatment of no ND with 12.8 MPa of US pressure, the second column is the PFP NDs mediated histotripsy with 12.8 MPa of US pressure, the third column is no ND with 26.2 MPa of US pressure, and the forth column is the PFH NDs mediated histotripsy with 12.8 MPa of US pressure. Each row is separated with before, during, and after US treatments. The spheroid border, cavitation bubbles, and ablated spheroid section were encircled with red, white, and yellow colors, respectively to be easily distinguished the treatment efficiency. The cross bar is 200 µm. (D) Calculation of the fractionated spheroid area after the ablation treatment with low pressure (12.8 MPa), high pressure (26.2 MPa), PFP loaded NDs, or PFH loaded NDs. The areas are calculated from the ultrafast recording camera images equipped with a 4X microscope objective. Results are the average of three independent experiments ± standard error of the mean. Figure 8: (A) Regrown of the ablated PC-3 cells with PFH-NDs under US for 2 days comparing to untreated spheroids under light microscopy (the cross-bar shows 200 µm) and (B) assayed with resazurin viability dye.

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Figure 1. 32


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Figure 2.

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A

B

Figure 3. 34


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(A)

(B)

Figure 4

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(A)

(B)

Figure 5.

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(A)

(B)

Figure 6.

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(A)

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(B)

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(C)

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(D)

Figure 7

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(A)

(B)

Figure 8.

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Table of Content Figure. Schematic drawing of prostate cancer spheroids and images of PC-3 and C4-2B spheroids acquired using light microscope (i and iii) or fluorescence microscope (ii and iv) after staining with Calcein AM dye showing live cancer cells (green). Treatment of prostate cancer spheroids with PFP or PFH-loaded NDs and application of therapeutic ultrasound results in rapid formation and collapse of microbubbles that mechanically fractionate the cancer cells.

43


Noninvasive Ablation of Prostate Cancer Spheroids Using Acoustically

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