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

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

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20–25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reductionThis work was supported by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed III; by Consellería de Educación e Ordenación Universitaria, Xunta de Galicia's Grupos de referencia competitiva (grant number ED431C 2017/09). The authors thank TÜBİTAK (The Scientific and Technical Research Council of Turkey) for supporting this project (Project number : 217S068). S.A acknowledges the financial support for his postdoctoral research by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed IIIS

Nanomedicine and Sonopermeation in the Treatment of Cancer

Treatment of cancer is an enormous challenge that involves multiple biological barriers and drugs with limited specificity, which makes treatment both ineffective and debilitating to the patient. One of the approaches that have gotten attention during the last three decades is encapsulation of drugs into nanoparticles, to create nanomedicines. Nanoparticles can protect drugs from degradation and protein binding, alter the biodistribution to protect healthy tissue from the drug, and to some degree accumulate in tumors due to the enhanced permeability and retention effect. However, due to the size of nanomedicines, the transport barriers in tumors are even greater challenges for nanomedicines than for molecular drugs. The blood brain barrier effectively stops entry into tumors in the brain, and the dense extracellular matrix, interstitial pressure and solid stress in tumors elsewhere hinders transport of the nanoparticles. Ultrasound in combination with microbubbles can be used for what we have described as sonopermeation, which can improve the effect of drugs and nanomedicines both in the brain and in solid tumors. The mechanisms behind the improved effect are not fully understood. In this thesis the development of a drug delivery product consisting of nanoparticles and nanoparticle-stabilized microbubbles to be used for sonopermeation is described. The nanoparticles are made by miniemulsion synthesis of poly(alkyl cyanoacrylate). In the first part of the thesis these nanoparticles are characterized for cellular uptake, intracellular degradation and drug release, surface properties and toxicity, which are all important properties of a drug delivery system. We found that poly(ethyl-buthyl cyanoacrylate) nanoparticles covered with two relatively short PEGs had the most favorable properties and were used in subsequent studies. The second part of this thesis revolves tumor models and the effect of sonopermeation with the nanoparticle-microbubble system. We evaluated five tumor models for nanoparticle-related properties and found that blood vessel quantity and quality were both important factors. We found that the effect of sonopermeation differed between two of them amongst other due to the solid stress in the tumor. We also used the nanoparticle-microbubble system to open the blood brain barrier in an orthotopic glioma model in mice, and found that we could permeate the blood brain barrier, but that this was not sufficient to achieve improved drug delivery probably due to the presence of efflux pumps. This thesis shows that the development of nanomedicine is challenging. This is both due to technicalities of producing nanoparticles with a defined set of properties, and due to the biological barriers that differ between patients and diseases. The thesis is concluded by an opinon-paper where we suggest that a more disease-driven approach to nanomedicine is needed, but that nanomedicine will play an important role in future treatment regimens amongst other by using the nanomedicine toolbox in combination with external forces such as ultrasound

Mechanisms of Cellular Uptake and Intracellular Degradation of Polymeric Nanoparticles

The effect of cancer therapy could be greatly improved by encapsulating existing drugs into nanoparticles. Nanoparticles can facilitate delivery of hydrophobic drugs with poor solubility in water, and allow you to target cancer cells specifically either through passive targeting, active targeting or triggered drug delivery. This thesis explores the cellular uptake and intracellular degradation of a novel, multimodal polymeric nanoparticle developed at SINTEF Materials and Chemistry. Two types of nanoparticles were evaluated for drug delivery purposes. Fluorescence lifetime imaging and analysis of emission spectra were used to assess the intracellular degradation, confocal laser scanning microscopy and flow cytometry were used to characterized the uptake.It was found that the poly(butyl cyanoacrylate) particle has degraded considerably after 24 hours intracellularly and is taken up through clathrin-mediated endocytosis. It was found that the cellular uptake was highly dependent on cell confluency and maturation. The poly (octyl cyanoacrylate) nanoparticle was not found to degrade readily within the first week of cell contact and is therefore less promising for drug delivery purposes. However, it was found that after 3 hours the particle had at least 3-fold higher uptake in prostate cancer cells than the poly(butyl cyanoacrylate) particle and that both clathrin- and caveolin-mediated uptake was important mechanisms in this uptake. This particle might be useful either for slow delivery or imaging. It was found indications that the nanoparticles escape the lysosomes and degrades in cytosol which is beneficial for drug delivery

Extracellular vesicles in patients in the acute phase of psychosis and after clinical improvement: an explorative study

Extracellular vesicles (EVs) are cell-derived structures that transport proteins, lipids and nucleic acids between cells, thereby affecting the phenotype of the recipient cell. As the content of EVs reflects the status of the originating cell, EVs can have potential as biomarkers. Identifying EVs, including their cells of origin and their cargo, may provide insights in the pathophysiology of psychosis. Here, we present an in-depth analysis and proteomics of EVs from peripheral blood in patients (n = 25) during and after the acute phase of psychosis. Concentration and protein content of EVs in psychotic patients were twofold higher than in 25 age- and sex-matched healthy controls (p < 0.001 for both concentration and protein content), and the diameter of EVs was larger in patients (p = 0.02). Properties of EVs did not differ significantly in blood sampled during and after the acute psychotic episode. Proteomic analyses on isolated EVs from individual patients revealed 1,853 proteins, whereof 45 were brain-elevated proteins. Of these, five proteins involved in regulation of plasticity of glutamatergic synapses were significantly different in psychotic patients compared to controls; neurogranin (NRGN), neuron-specific calcium-binding protein hippocalcin (HPCA), kalirin (KALRN), beta-adducin (ADD2) and ankyrin-2 (ANK2). To summarize, our results show that peripheral EVs in psychotic patients are different from those in healthy controls and point at alterations on the glutamatergic system. We suggest that EVs allow investigation of blood-borne brain-originating biological material and that their role as biomarkers in patients with psychotic disorders is worthy of further exploration.publishedVersio

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

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 suchnas 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

Therapeutic Effect of Cabazitaxel and Blood-Brain Barrier opening in a Patient-Derived Glioblastoma Model

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor. Surprisingly, neither ultrasound-mediated blood-brain barrier opening (sonopermeation) nor drug formulation in polymeric nanoparticles could increase either accumulation of the drug in the brain or therapeutic effect. This indicates that for hydrophobic drugs, sonopermeation of the blood brain barrier might not be sufficient to achieve improved drug delivery. Nonetheless, our study shows that cabazitaxel is a promising drug for the treatment of brain tumors.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