Integrating Therapeutic Ultrasound With Nanosized Drug Delivery Systems in the Battle Against Cancer

Abstract

Controlled, localized, and timely activation of nanosized drug delivery systems (NSDDSs), using an external stimulus such as therapeutic ultrasound (TUS), can improve the efficacy of cancer treatments compared to either conventional chemotherapy methods or passive NSDDSs alone. Specifically, TUS induces thermal and mechanical effects that trigger drug release from NSDDSs and overcomes drug delivery barriers in tumor microenvironments to allow nanoparticle drug carriers to penetrate more deeply into tumor tissue while minimizing side effects. This review highlights recent advancements, contemplates future prospects, and addresses challenges in using TUS-mediated NSDDSs for cancer treatment, encompassing preclinical and clinical applications.

Introduction

The integration of nanosized drug delivery systems (NSDDSs) with therapeutic ultrasound (TUS) has significant potential to improve the efficacy of cancer therapies. ¹ Various nanoparticle drug carriers have been designed to enter the tumor vasculature and achieve higher intratumoral accumulation via a phenomenon known as the enhanced permeability and retention (EPR) effect. Ultrasound is a complementary modality that can penetrate tissue more than an order of magnitude deeper than near-infrared light, and therefore readily interacts with various types of drug-loaded nanoparticles located deep in the circulatory system, tumor capillary network, and extracellular matrix (ECM). Ultrasound parameters such as pressure, frequency, spatial profile, and exposure time can be tuned to trigger on-demand drug release from different NSDDSs. In combination, TUS and NSDDSs can provide an attractive alternative to conventional chemotherapeutic drug delivery options because they combine the deep penetration ability of TUS with the on-demand capability of TUS-induced drug release of NSDDSs to localize the delivery of therapeutic agents into the region of interest (ROI). ² Furthermore, the localized drug delivery achieved at the tumor site can minimize side effects^3,4 and address challenges associated with NSDDSs, which include (i) poor uptake and accumulation of nanoparticles by cells; (ii) a limited amount of drug released from nanoparticles; and (iii) low efficiency of localized nanoparticle accumulation via the EPR effect.

This mini-review provides an overview of the latest advancements in TUS-mediated NSDDSs as an emerging and site-specific approach for cancer treatment. In addition, we outline preclinical in silico computational, ex vivo tissue, in vitro cell culture, and in vivo animal models as well as different clinical practices that shed light on potential safety considerations. Finally, we discuss the practical challenges and future prospects of this technology.

Therapeutic Ultrasound Applications in NSDDSs

The applications of TUS are often limited to soft tissues, with limitations in using TUS for hard tissues (eg, bones) or air-filled tissues (eg, lungs and digestive system). More specifically, TUS is directed locally in areas where blood vessels and the ECM play crucial roles, because TUS can increase vascular permeability and thereby enhance drug extravasation and delivery to targeted tissues. For tumor treatment, TUS can propagate through the tumor's rigid and dense ECM that otherwise limits nanoparticle and drug penetration. ⁵ In addition, TUS induces mechanical forces that can potentially push drugs or nanoparticles deeper into the tissue. Tuning TUS to optimize these outcomes requires a thorough understanding of the effects of TUS on blood vessels and the ECM.

Physiological barriers such as the endothelium, tumor microenvironment, and immunological barriers impede efficient drug delivery to tumors for 2 main reasons ⁴ : (i) large-molecule therapeutics cannot efficiently enter the narrow fenestrae within crosslinked matrix components in the ECM; and (ii) the combination of a dense ECM and limited lymphatic tumor drainage increases the interstitial fluid pressure that inhibits the infiltration of immune cells and the convection of small drug agents. Endothelial cells pose a significant barrier for intravenously administered drugs since the majority of drug targets are situated outside the blood vessels. Nanoparticles must traverse this endothelium, characterized by tight junctions between endothelial cells. Pioneering research has shown that ultrasound can stimulate the release of several signaling molecules, increasing the permeability of blood vessels and cell membranes.^1,4,6,7

In TUS-mediated NSDDSs, TUS interacts with both tissue and nanoparticles, thereby enhancing the delivery of therapeutic agents to specific tissues or an ROI within the body. TUS generates high-frequency vibrations causing mechanical effects such as microstreaming and cavitation in tissues while interacting with nanoparticles by inducing a controlled cavitation around them. This cavitation can improve the dispersion and interaction of nanoparticles with the surrounding tissue, allowing for a more efficient therapy. In addition, tissue and nanoparticles both absorb and convert TUS energy into heat. Localized hyperthermia induced by acoustic field drives the energy conversion of TUS into heat, thereby enhancing drug release from nanoparticles and improving drug diffusion in tissue. TUS-nanoparticle interaction generally involves a combination of mechanical agitation and controlled heating, leading to enhanced drug release and targeted delivery.^1,4,6 In addition, various types of ultrasound-sensitive nanoparticles can be designed to enable controlled drug release or other therapeutic effects in the ROI. Various nanoplatforms are available for developing target-specific TUS-activated NSDDSs, which can be distinguished based on their chemistry and biodegradability. ⁸ Examples include lipid-based nanosystems (eg, liposomes and nanoemulsions), micro/nanobubbles, dendrimers, proteins, polymers, and nanomaterials with inorganic, organic, and metal structures. ⁶ These materials are susceptible to thermal and nonthermal interactions caused by TUS. In the case of targeting tissues, TUS can induce a combination of thermal and nonthermal interactions in tissue, depending on its pressure amplitude, frequency, and duty cycle. Increasing thermal and nonthermal TUS interactions allows higher drug release from NSDDSs, improving drug diffusion within the tissue and thereby enhancing overall therapeutic outcomes. Nonthermal interactions include acoustic radiation force, acoustic streaming, and the formation and cavitation of microbubbles and nanobubbles. ⁴ Thermal interactions arise from the transformation of acoustic energy to thermal energy, resulting in a volumetric temperature rise in the ROI.^2,4,7,9 Acoustic streaming occurs for a large range of ultrasound intensities, while acoustic cavitation typically occurs at high intensities when using TUS exposures with high mechanical indexes. ¹⁰

Therapeutic ultrasound typically incorporates one of 3 noninvasive approaches for drug delivery, specifically focusing on drug release triggered by the ultrasound field: low-intensity pulsed ultrasound (LIPUS), low-intensity focused ultrasound (LIFU), and high-intensity focused ultrasound (HIFU). Unfocused ultrasound (LIPUS) treats large ROIs (eg, a few centimeters), while focused ultrasound (LIFU and HIFU) treats small ROIs (eg, a few millimeters). ¹¹ All 3 approaches have been used to induce drug release from drug-loaded nanoparticles. ⁴ Parameters such as transducer power, ultrasound pressure, exposure time, and duty cycle should be optimized to limit the maximum tissue temperature to 44 °C (or a maximum thermal dose, ¹¹ as an upper limit of localized moderate hyperthermia), thus minimizing the risk of thermal damage. This is achieved in clinical applications using a temperature controller that maintains a tissue temperature below 44 °C (or thermal dose) during the treatment (eg, magnetic-resonance guided focused ultrasound [MRgFUS]). ¹² However, if the timeframe is short enough, the transducer power and duty cycle can be tuned to limit the temperature increase without a temperature controller. ¹³ In a recent study, a combination of nonthermal and thermal interactions caused by LIPUS was found to be responsible for drug release from the surface of gold nanoparticles. ¹⁴ Therefore, since the contributions of nonthermal and thermal interactions could vary between LIPUS, LIFU, and HIFU, the appropriate approach depends significantly on the type of drug or carrier used. For example, thermosensitive liposomes are more suitable than cavitation-sensitive liposomes if thermal interactions dominate and vice versa.

While propagating in tissue, ultrasound attenuates and deposits its energy into heat. During thermal therapy of cancerous tissue, heat generation induces hyperthermia ¹⁵ at elevated temperatures (< 44 °C) or thermal ablation that causes irreversible cell damage at even higher temperatures.^16,17 An increase in temperature affects normal tissue and cancerous tissue differently. For example, in normal tissue, hyperthermia causes momentary blood vessel dilation, a mild increase in blood flow, and triggers a heat-shock response aimed at protecting the cell from thermal injury and repairing any resulting damage.^18,19 Conversely, tumor blood vessels are usually at near maximal dilation, so no significant change to blood flow occurs during moderate hyperthermia (eg, between 40 °C and 42 °C).^18,19 Increasing the temperature above 44 °C initiates an irreversible reduction in tumor blood flow, causing vascular stasis and thrombosis that ultimately results in heat-trapping. ¹⁹ This tumor flow decrease will also decrease tumor pO₂ and further decreases the pH of a tumor's naturally acidic microenvironment. In contrast, the pH of normal vasculature is unchanged by hyperthermia due to tightly regulated physiological processes. In both normal and tumor tissue, thermal ablation (> 44 °C) inactivates proteins and enzymes at the cellular level and can cause cell death. Overall, the impact of heat on cancer cells depends on temperature, duration of exposure, tumor microenvironment, and tumor biology. Hyperthermia can improve the delivery of drug-loaded nanoparticles into a tumor by enhancing blood vessel permeability and promoting nanoparticle penetration, while enhanced drug release from NSDDSs can occur via increased diffusion rates and improved nanoparticle degradation. ¹⁷ Delivery of ultrasound-induced hyperthermia, with a compatible modality (such as chemotherapy), should be customized to align with specific treatment objectives. ⁴

Preclinical and Clinical Trials

Different types of cancer cells will respond differently to TUS-activated NSDDSs. Both preclinical (in silico, ex vivo, in vitro, and in vivo) and human clinical trials on various cancer cell lines and tumor types are conducted in many research labs and medical hospitals to understand the effect of TUS-activated NSDDSs. In recent studies, in silico approaches have been investigated in TUS (mostly focused ultrasound [FUS])-activated NSDDSs,^14,20–22 while ex vivo trials for unfocused TUS (ie, LIPUS)-triggered drug release from gold nanoparticles have also been investigated.^13,14 Multiple cell lines, involving leukemia, breast cancer, colon adenocarcinoma, ovarian carcinoma, and colorectal carcinoma have also been studied in in vitro preclinical experiments.^1,4,23 In addition, in vivo preclinical trials have been carried out on various cancer types, such as pancreatic, breast, colon, lymphoma, prostate, and ovarian cancers.^1,4,23 These studies have explored the influence of both mechanical and thermal effects induced by LIFU, HIFU, FUS, and LIPUS. ⁴ A TUS-activated NSDDS using thermosensitive liposomes successfully treated liver^24,25 and pancreatic^26,27 malignancies in human clinical trials. Lyon et al^24,25 introduced the first human proof of concept for a FUS-triggered NSDDS, termed TARDOX. This approach aimed to enhance ThermoDox® delivery with localized hyperthermia using FUS for liver tumors; ten patients were treated. Results demonstrated a significant increase in the intratumoral concentration of doxorubicin (DOX). Based on these studies, the group later carried out a Phase I PanDox study, aimed to enhance DOX uptake in pancreatic tumors via ThermoDox® and FUS. ²⁶

In practice, the anatomical location of the target tumor in the human body, which contains skin, solid bones, gas-containing organs, the blood circulatory system, and nervous and respiration systems, can limit TUS propagation and penetration due to ultrasound attenuation and reflection. ⁴ For example, compared with soft tissue, solid bones have a higher density that causes significant ultrasound attenuation and absorption, leading to heating and pain. ²⁸ In addition, the acoustic impedance mismatch between soft tissue and bone and between body fluids and gas-containing organs can lead to significant reflection, scattering, and absorption of ultrasound waves, preventing sufficient acoustic energy deposition at the target to trigger drug release or therapeutic effects. Furthermore, real-time image guidance of the TUS target region is necessary to ensure that any voluntary or involuntary body movements (eg, breathing and cardiac motion) do not significantly shift the target (eg, ROI or tumor) during treatment. As an example, magnetic resonance imaging has been combined with TUS in image-guided focused ultrasound (eg, MRgFUS) to successfully target various tumor types (eg, breast, bone, and liver) as well as to open the blood–brain barrier (BBB) to allow nanoparticle drug carrier access to brain tumors. ²⁸ Specific types of transducers, like phased array TUS transducers with large surface areas, significantly limit skull hot spots and provide sharp focal areas in MRgFUS brain surgery, while low frequencies (600-700 kHz) allow the passage of ultrasound through the skull for noninvasive thermal ablation of brain tumors. ²⁸ For a given tumor type, acoustic interactions with the skin, tissue, and bone layers preceding the tumor must be carefully evaluated in the planning stage to optimize the ultrasound propagation during treatment.

In the human body, diagnostic ultrasound applications must meet specific thermal and mechanical index thresholds to reduce undesirable risks in operation, as outlined by the Food and Drug Administration (FDA) and the British Medical Ultrasound Society. The thermal index provides information about the potential for heating of tissues due to the ultrasound and depends on the time-averaged acoustic power, the acoustic properties of tissue and blood flow in the ROI, and the path of the ultrasound beam. Ultimately, the ROI should not experience a temperature increase of more than 6 °C above the normal body temperature.^29,30 In addition, the mechanical index must fall below 0.7 or 1.9, depending on whether the ultrasound encounters preexisting gas pockets or not, respectively.^4,7,9,29

Discussion and Future Directions

In contrast to diagnostic ultrasound, which has well-established guidelines, the utilization of TUS in conjunction with NSDDSs for cancer treatment lacks specific published recommendations, despite recent advancements in this field. Consequently, when integrating TUS with NSDDSs, a comprehensive assessment of potential hazards, side effects, therapeutic efficacy, and overall outcomes is required. Specifically, the long- and short-term thermal, mechanical, biological, and chemical effects induced by TUS at the cellular and tissue levels must be evaluated to establish optimized TUS parameters when planning clinical human trials. Furthermore, careful evaluation of the drug release kinetics from carriers in various NSDDSs, the duration of ultrasound exposure in different tumor environments (extracellular, intravascular, and intracellular), and the mode of nanoparticle administration (eg, intravenous, intratumoral, and intraperitoneal) is essential to ascertain the risk-to-benefit ratio effectively.^4,20

For biomedical TUS applications, emerging nano-structures require stringent and comprehensive nano-toxicity assessments on human health, including (i) the interaction of nanoparticles with different tissues/organs, including the central nervous system; (ii) nanoparticle toxicity; (iii) nanoparticle metabolism and accumulation in different tissues/organs; (iv) accidental overdose; and (v) long-term side effects. To improve the success rate of anti-cancer NSDDSs towards clinical translation, it is important to develop a stable, long-circulating drug delivery nanocarrier system with a targeted and timely drug release. Furthermore, several practical factors for NSDDSs, such as formulation cost, storage conditions, shelf life, time, and resources needed in research and development, serve as constraints on the clinical implementation of TUS-responsive NSDDSs. Achieving clinical translation also requires large-scale production of nanocarriers with high yield and consistency. This large-scale production will inevitably become an intricate and multistep process that will hinder the incorporation of nanocarriers into the market. While small batches are manageable for optimization and research purposes, scaling up production and ensuring quality control would require the substantial expense of designing new processes for the formulation scale-up. In addition to manufacturing costs, substantial expenses and time are involved in preclinical research, development, and human clinical trials.

Although numerous nanomedicines have been tested in recent clinical trials, only a small fraction have been approved for human clinical trials. The success of clinical translation of TUS-responsive nano-formulations for cancer therapy depends on sophisticated state-of-the-art designs and approaches, alongside a comprehensive understanding of the interactions between NSDDSs and organs/tissues/cells. To facilitate the bench-to-bed translation of this technology, comprehensive investigations are necessary to understand the complex interactions of NSDDS in vivo. Through meticulous in silico, in vitro, and in vivo research, a comprehensive understanding of nanocarrier biodistribution, aggregation, and stability can be established, leading to significant advancements and knowledge in this field. Sophisticated spatial–temporal multiscale and multiphysics computational models can be employed to explore the challenges in delivering anti-cancer agents within the complex and heterogeneous tumor microenvironment.^20,22

Computational/artificial intelligence models can aid personalized medicine by considering patient-specific factors to tailor treatment strategies. Computational models in TUS-mediated NSDDS provide valuable insights, guiding the design and implementation of both the preclinical and clinical stages. They can simulate different scenarios and predict outcomes for individual patients, optimizing ultrasound parameters and nanoparticle formulations for maximum therapeutic benefit with minimal side effects. The multiscale and multiphysics modeling of TUS-triggered NSDDSs encompasses multiple stages and principles, including the characterization of the acoustic wave field, drug release from nanoparticles through either thermal or nonthermal responses induced by TUS, interstitial fluid flow, drug transport within various spaces, and drug delivery to cells using pharmacodynamics models.^20,22 The Pennes bioheat transfer equation can be used to model the temperature distribution within the target area to simulate drug release from nanoparticles when TUS-induced heat is significant. Conversely, if nonthermal effects of TUS drive drug release, the model integrates acoustic streaming, acoustic radiation force, and cavitation, with drug release correlated to the acoustic pressure/intensity profile. ²⁰ On the other hand, machine learning algorithms may play a pivotal role in analyzing intricate datasets and identifying patterns to improve predictions and optimize various parameters. This approach contributes to the development of personalized treatment strategies by leveraging patient-specific data. These algorithms also aid in the creation of comprehensive and precise computational models that encompass the intricate interactions among nanoparticles, ultrasound waves, and biological tissues.

The main goal of ultrasound-mediated NSDDSs is to deliver an ultrasound field that propagates deep enough into tumor tissue to timely and locally trigger drug release from NSDDSs while minimizing damage to off-target cells and tissues. Promising avenues for future research in ultrasound-mediated NSDDSs include increasing the penetration depth of drugs using TUS-activated NSDDSs, in-depth studies on drug release kinetics from nanoparticles in response to different TUS approaches, and examining the effects of TUS and nanomedicine on the immune system and cellular uptake. In addition, a promising opportunity lies in the simultaneous utilization of NSDDSs with real-time imaging techniques (eg, MRgFUS) for monitoring purposes. In-depth research is required with various models (eg, in silico, ex vivo, in vitro, and in vivo) at multiple scales (macro → micro → nano) to enhance understanding of (i) the TUS interaction with tissues, cells, and nanoparticles; (ii) the nanoparticle drug release kinetics, (iii) drug transport in microvessels and tissue, and eventually (iv) drug delivery into cells toward improving therapeutic outcomes. Clinical translation of TUS-activated NSDDSs may soon be a breakthrough in nanomedicine by combining ex vivo, in vitro, and in vivo tests with novel imaging platforms and optimizing the process using multiscale and multiphysics computational modeling and machine learning.