Ultrasound and microbubbles to beat barriers in tumors: Improving delivery of nanomedicine

Successful delivery of drugs and nanomedicine to tumors requires a functional vascular network, extravasation across the capillary wall, penetration through the extracellular matrix, and cellular uptake. Nanomedicine has many merits, but penetration deep into the tumor interstitium remains a challenge. Failure of cancer treatment can be caused by insufficient delivery of the therapeutic agents. After intra-venous administration, nanomedicines are often found in off-target organs and the tumor extracellular matrix close to the capillary wall. With circulating microbubbles, ultrasound exposure focused toward the tumor shows great promise in improving the delivery of therapeutic agents. In this review, we address the impact of focused ultrasound and microbubbles to overcome barriers for drug delivery such as perfusion, extravasation, and transport through the extracellular matrix. Furthermore, we discuss the induction of an immune response with ultrasound and delivery of immuno-therapeutics. The review dis-cusses mainly preclinical results and ends with a summary of ongoing clinical trials.publishedVersio

Identification of novel cyanoacrylate monomers for use in nanoparticle drug delivery systems prepared by miniemulsion polymerisation – A multistep screening approach

Poly (alkyl cyanoacrylate) (PACA) polymeric nanoparticles (NPs) are promising drug carriers in drug delivery. However, the selection of commercially available alkyl cyanoacrylate (ACA) monomers is limited, because most monomers were designed for use in medical and industrial glues and later repurposed for drug encapsulation. This study therefore aimed to seek out novel ACA materials for use in NP systems using a toxicity led screening approach. A multistep strategy, including cytotoxicity screening of alcohols as degradation products of PACA (44 alcohols), NPs (14 polymers), and a final in vivo study (2 polymers) gave poly (2-ethylhexyl cyanoacrylate) PEHCA as a promising novel PACA candidate. For the first time, this work presents cytotoxicity data on several novel ACAs, PEHCA in vivo toxicity data, and miniemulsion polymerisation-based encapsulation of the cabazitaxel and NR688 in novel PACA candidates. Furthermore, several of the ACA candidates were compatible with a wider selection of lipophilic active pharmaceutical ingredients (APIs) versus commercially available controls. Combined, this work demonstrates the potential benefits of expanding the array of available ACA materials in drug delivery. Novel ACAs have the potential to encapsulate a wider range of APIs in miniemulsion polymerisation processes and may also broaden PACA applicability in other fields.publishedVersio

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.publishedVersio

Formulation and characterisation of drug-loaded antibubbles for image-guided and ultrasound-triggered drug delivery

The aim of this study was to develop high load-capacity antibubbles that can be visualized using diagnostic ultrasound and the encapsulated drug can be released and delivered using clinically translatable ultrasound. The antibubbles were developed by optimising a silica nanoparticle stabilised double emulsion template. We produced an emulsion with a mean size diameter of 4.23 ± 1.63 µm where 38.9 ± 3.1% of the droplets contained a one or more cores. Following conversion to antibubbles, the mean size decreased to 2.96 ± 1.94 µm where 99% of antibubbles were <10 µm. The antibubbles had a peak attenuation of 4.8 dB/cm at 3.0 MHz at a concentration of 200 × 103 particles/mL and showed distinct attenuation spikes at frequencies between 5.5 and 13.5 MHz. No increase in subharmonic response was observed for the antibubbles in contrast to SonoVue®. High-speed imaging revealed that antibubbles can release their cores at MIs of 0.6. In vivo imaging indicated that the antibubbles have a long half-life of 68.49 s vs. 40.02 s for SonoVue®. The antibubbles could be visualised using diagnostic ultrasound and could be disrupted at MIs of ≥0.6. The in vitro drug delivery results showed that antibubbles can significantly improve drug delivery (p < 0.0001) and deliver the drug within the antibubbles. In conclusion antibubbles are a viable concept for ultrasound guided drug delivery.publishedVersio

Quantification and qualitative effects of different PEGylations on Poly(butyl cyanoacrylate) nanoparticles

Protein adsorption on nanoparticles (NPs) used in nanomedicine leads to opsonization and activation of the complement system in blood, which substantially reduces the blood circulation time of NPs. The most commonly used method to avoid protein adsorption, is to coat the NPs with polyethylene glycol, so called PEGylation. Although PEGylation is of utmost importance for designing the in vivo behavior of the NP, there is still a considerable lack of methods for characterization and fundamental understanding related to the PEGylation of NPs. In this work we have studied four different poly(butyl cyanoacrylate) (PBCA) NPs , PEGylated with different types of PEG-based non-ionic surfactants–Jeffamine M-2070, Brij L23, Kolliphor HS 15, Pluronic F68–or combinations thereof. We evaluated the PEGylation, both quantitatively by nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), and qualitatively by studying zeta-potential, protein adsorption, diffusion, cellular interactions and blood circulation half-life. We found that NMR and ToF-SIMS are complementary methods, while TGA is less suitable to quantitate PEG on polymeric NPs. It was found that longer PEG increases both blood circulation time and diffusion of NPs in collagen gels

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects

Ultrasound-mediated delivery of nanomedicine across biological barriers - for improved treatment of cancer and diseases in the brain

Despite major progress in the field of cancer research during the last decades, cancer remains a leading cause of death worldwide. Treatment with conventional chemotherapy is limited by inadequate delivery to the tumor and severe side-effects due to accumulation in healthy tissues. Encapsulation of drugs in nanoparticles can enable a more targeted delivery, for improved efficacy and reduced toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous, due to various biological barriers in the tumor microenvironment. Ultrasound in combination with microbubbles has emerged as a promising method to enhance delivery of nanomedicines to tumor tissue. It can also facilitate non-invasive and localized opening of the blood-brain barrier for drug delivery to the brain. A unique microbubble-platform was investigated, which demonstrated great potential for controlled drug delivery. It consisted of a shell of polymeric nanoparticles, encapsulating drugs and contrast agents. Various payloads demonstrated different degree of stability in the nanoparticles, and also affected nanoparticle-cell interactions. Depending on the payload, the nanoparticles could effectively deliver hydrophobic dyes and drugs to cells by either contact-mediated delivery directly to cytosol, or uptake by endocytosis followed by intracellular release. Cellular uptake and toxicity of the nanoparticles, as well as in vivo circulation and biodistribution were determined. Ultrasound treatment caused cavitation and collapse of the microbubbles, resulting in significantly improved accumulation and distribution of nanoparticles in solid tumors. Too low pressures, or short pulses did not enhance uptake, whereas too high pressures combined with long pulses resulted in tissue damage. In a proof-of-principle therapy study, the enhanced delivery resulted in complete and stable remission in all animals. Furthermore, it was shown that this platform could be used in combination with ultrasound to open the blood-brain barrier and successfully deliver and distribute nanoparticles in the brain. Another highly interesting microbubble-platform was also explored, based on clusters of microdroplets and microbubbles, which upon activation by ultrasound form large bubbles that deposit in the microvasculature. They were previously shown to enhance therapeutic efficacy of co-injected drugs to tumors in mice. Herein, we demonstrated that the same platform could be used to transiently and safely open the blood-brain barrier for delivery of macromolecules to the brain. These results provide an important fundament for future studies, and indicate that ultrasound-mediated delivery of nanomedicines across biological barriers can significantly improve treatment of cancer and diseases in the brain

Real-Time Multiphoton Intravital Microscopy of Drug Extravasation in Tumours during Acoustic Cluster Therapy

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5–10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT’s mechanisms and potential in localised drug delivery

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.publishedVersio

Ultrasound-Mediated Delivery of Chemotherapy into the Transgenic Adenocarcinoma of the Mouse Prostate Model

Ultrasound (US) in combination with microbubbles (MB) has had promising results in improving delivery of chemotherapeutic agents. However, most studies are done in immunodeficient mice with xenografted tumors. We used two phenotypes of the spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model to evaluate if US + MB could enhance the therapeutic efficacy of cabazitaxel (Cab). Cab was either injected intravenously as free drug or encapsulated into nanoparticles. In both cases, Cab transiently reduced tumor and prostate volume in the TRAMP model. No additional therapeutic efficacy was observed combining Cab with US + MB, except for one tumor. Additionally, histology grading and immunostaining of Ki67 did not reveal differences between treatment groups. Mass spectrometry revealed that nanoparticle encapsulation of Cab increased the circulation time and enhanced the accumulation in liver and spleen compared with free Cab. The therapeutic results in this spontaneous, clinically relevant tumor model differ from the improved therapeutic response observed in xenografts combining US + MB and chemotherapy.publishedVersio