Novel Ultrasound-Responsive Amyloid Formulation.

Amyloid aggregates have attracted significant interest in regard to diverse biomedical applications, particularly in the field of drug delivery. Here, we report novel amyloid aggregates based on a 12-amino-acid peptide from the amyloidogenic region of the receptor-interacting kinase 3 (RIP3) protein and a thermoresponsive triblock copolymer, namely, Pluronic F127 (RIP3/F127). Physicochemical characterization was performed to determine the aggregation size, morphology, and stimuli-responsive properties. The potential of the aggregates as a drug depot was assessed in lung cancer cells, using Doxorubicin (Dox) as a model drug. The results show that RIP3 and RIP3/F127 exhibit amyloidogenic properties. Further, the RIP3/F127 amyloids exhibited significant ultrasound-responsive properties compared to amyloid aggregates without Pluronic F127. Moreover, the RIP3/F127/Dox amyloid formulations that were subjected to ultrasound treatment exhibited greater toxicity to lung cancer cells compared to that of Dox alone at equal concentrations. Overall, the results from this proof-of-concept study show that amyloidogenic peptide aggregates with stimuli-responsive properties can be utilized as efficient drug delivery depots.

Effect of extracellular matrices on production and potency of mesenchymal stem cell-derived exosomes.

Mesenchymal stem cell (MSC) derived exosomes have emerged as potential acellular therapeutics for various tissue regenerative applications. However, successful clinical translation of exosome-based therapy is limited by lack of a structured production platform. Thus, in this study, the effect of decellularized extracellular matrix (dECM) was assessed on the production and potency of exosomes secreted by bone marrow-derived human MSCs. The results indicate that there was a ∼2-fold increase in MSC-exosome production when MSCs were cultured on dECM compared to TCP. Further, our study revealed that dECM generation induced by ascorbic acid (AA) up to 100 µg mL-1 highly increased exosome yield thereby indicating a potential scale up method for MSC exosome production. The bioactivity of exosomes was investigated by their ability to improve the healing of wounded human skin explants. Wound closure was enhanced in the presence of exosomes isolated from MSCs cultured on ascorbic acid-induced dECM compared to TCP generated MSC-exosomes. In summary, this study suggests a promising solution to a major bottleneck in large-scale production of MSC exosomes for cell-free therapy.

Amyloid-like RIP1/RIP3 RHIM Fragments' Characterization and Application as a Drug Depot.

Amyloid aggregates play a major role in diseases as well as in normal physiological function. Receptor-interacting protein kinases 1 and 3 (RIP1/RIP3) aggregates complexes in cellular necroptosis is one example of protein aggregation in normal cellular function. Although recently there have been several studies on full kinase proteins aggregation, the aggregation potential of small peptide sequences of RIP1/RIP3, the physicochemical properties, and the potential in biomedical applications have not been explored. Hence, in this paper, we study the aggregation propensity of peptides consisting of four and twelve amino acid sequences in the RHIM region of RIP1/RIP3 proteins that are known to drive the beta-sheet formation and the subsequent aggregation. The aggregation kinetics, physicochemical characterization, mechanosensitive properties, cellular effects, and potential as a cancer drug depot have been investigated. The results show that the number and concentration of amino acids play a role in amyloid-like aggregates' properties. Further, the aggregates when formulated with cisplatin-induced significant lung cancer cell toxicity compared to an equal amount of cisplatin with and without ultrasound. The study would serve as a platform for further investigation on RIP1/RIP3 peptide and protein aggregates, their role in multiple cellular functions and diseases, and their potential as drug depots.

Exosome-Coated tPA/Catalase Nanoformulation for Thrombolytic Therapy.

Current tissue plasminogen-based therapeutic strategies for stroke suffer from systemic side effects and poor efficacy. Hence, novel drug delivery methods are needed to overcome these shortcomings. Exosome-based drug formulations have been shown to have superior therapeutic outcomes compared to conventional systemic drug delivery approaches. In this paper, we report exosome surface-coated tissue plasminogen activator (tPA)/catalase nanoformulations with improved thrombolytic efficacy compared to free tPA, which also reduce side effects. The results showed that the tPA exosome formulations retained tPA activity, improved tPA stability, exhibited significant fibrinolysis, and showed no significant toxicity effects. Further, when combined with antioxidant enzyme catalase, the formulation was able to inhibit hydrogen peroxide-mediated oxidative stress and toxicity. Hence, exosome-based tPA/catalase nanoformulations could have the potential to offer a safer and effective thrombolytic therapy.

Heparin and Arginine Based Plasmin Nanoformulation for Ischemic Stroke Therapy.

Ischemic stroke is the most common type of stroke and thrombolytic therapy is the only approved treatment. However, current thrombolytic therapy with tissue plasminogen activator (tPA) is often hampered by the increased risk of hemorrhage. Plasmin, a direct fibrinolytic, has a significantly superior hemostatic safety profile; however, if injected intravenously it becomes rapidly inactivated by anti-plasmin. Nanoformulations have been shown to increase drug stability and half-life and hence could be applied to increase the plasmin therapeutic efficacy. Here in this paper, we report a novel heparin and arginine-based plasmin nanoformulation that exhibits increased plasmin stability and efficacy. In vitro studies revealed significant plasmin stability in the presence of anti-plasmin and efficient fibrinolytic activity. In addition, these particles showed no significant toxicity or oxidative stress effects in human brain microvascular endothelial cells, and no significant blood brain barrier permeability. Further, in a mouse photothrombotic stroke model, plasmin nanoparticles exhibited significant efficacy in reducing stroke volume without overt intracerebral hemorrhage (ICH) compared to free plasmin treatment. The study shows the potential of a plasmin nanoformulation in ischemic stroke therapy.

Exosomes Based Geldanamycin Delivery to Cancer Cells with Increased Therapeutic Efficacy.

HSP90 has been shown to promote oncogenic functions and cell proliferation during tumor progression. Geldanamycin is a potent inhibitor of HSP90, however therapeutic effects are often hampered due to its extreme hydrophobicity and systemic toxicity. Hence targeted delivery strategies are needed to overcome these limitations and to improve the efficacy of the drug. Here we utilize a novel geldanamycin delivery approach by utilizing exosomes. For the study, exosomes were extracted from cancer cells, loaded with geldanamycin, and the therapeutic effects were tested in cancer cells. Results show that cancer cells derived exosomes exhibit specificity to cancer cells. Further exosomes loaded geldanamycin show several fold increase in efficacy compared to free geldanamycin. Findings indicate exosomal formulations could be used for extremely hydrophobic HSP90 inhibitor geldanamycin delivery for inhibiting cancer cell proliferation.

Effect of cellular polyanion mimetics on tau peptide aggregation.

Tau protein aggregation is believed to be one of the key drivers of Alzheimer's disease. The two hexapeptide amino acid sequences 306 VQIVYK311 and 275 VQIINK280 of the tau protein are responsible for aggregation, and subsequent functional loss leading to Alzheimer's progression. Hence, it is important to understand the factors that promote the self-aggregation of this tau peptide fragments. Cellular microenvironmental polyanions are known to play a major role in tau protein aggregation and loss of function. Previous studies mainly focused on the effect of heparin and heparan sulfate solution on the full tau protein aggregation. Here, we study the effect of cellular polyanion mimetics, glycosaminoglycans (GAGs) in solution and on surface to mimic intracellular and extracellular conditions, and cellular RNA on the aggregation of tau hexapeptide 306 VQIVYK311 , and mutant tau peptide 273 GKVQIIN_KLDL284 found frequently in taupathy. Characterization techniques used for the study include biochemical assays, electron microscopy, and spectroscopy techniques to investigate the aggregation vitro. Results show while all the GAGs in solution form play a role in tau peptides aggregation, heparin, chondroitin sulfate, and dermatan sulfate in particular significantly promote the tau peptides aggregation formation. On the other hand, surface-coated GAGs did not promote significant tau aggregation. As far as cellular RNA is concerned, results show significant increase in tau peptides aggregation and toxicity in neuroblastoma cells. The study on the tau peptides fragments aggregation in the presence of GAGs and cellular RNA could provide novel information on the tau aggregation mechanism and could open ways for developing cellular polyanions modulating therapeutics for treating Alzheimer's disease.

Treating p53 Mutant Aggregation-Associated Cancer.

p53 is a tumor suppressor protein. Under stressful conditions, p53 tightly regulates cell growth by promoting apoptosis and DNA repair. When p53 becomes mutated, it loses its function, resulting in abnormal cell proliferation and tumor progression. Depending on the p53 mutation, it has been shown to form aggregates leading to negative gain of function of the protein. p53 mutant associated aggregation has been observed in several cancer tissues and has been shown to promote tumor growth. Recent studies show correlation between p53 mutant aggregation, functional loss, and tumor growth. Moreover, p53 aggregation has been observed in biopsies, patient tissues, and in vivo studies. Given the fact that over fifty percent of cancers have p53 mutation and several of them are prone to aggregation, therapeutic strategies are needed for treating p53 mutant aggregation associated cancers. Recent studies using polyarginine analogues and designer peptides for inhibiting p53 aggregation and tumor growth gives further encouragement in treating cancer as a protein aggregation disease. In this review, we highlight the recent efforts in targeting p53 aggregation in cancer and propose the use of small stress molecules as potential p53-antiaggregation drugs.

Ultrasound-sensitive nanoparticle aggregates for targeted drug delivery.

Here we describe injectable, ultrasound (US)-responsive, nanoparticle aggregates (NPAs) that disintegrate into slow-release, nanoscale, drug delivery systems, which can be targeted to selective sites by applying low-energy US locally. We show that, unlike microbubble based drug carriers which may suffer from stability problems, the properties of mechanical activated NPAs, composed of polymer nanoparticles, can be tuned by properly adjusting the polymer molecular weight, the size of the nanoparticle precursors as well as the percentage of excipient utilized to hold the NPA together. We then apply this concept to practice by fabricating NPAs composed of nanoparticles loaded with Doxorubicin (Dox) and tested their ability to treat tumors via ultrasound activation. Mouse studies demonstrated significantly increased efficiency of tumor targeting of the US-activated NPAs compared to PLGA nanoparticle controls (with or without US applied) or intact NPAs. Importantly, when the Dox-loaded NPAs were injected and exposed to US energy locally, this increased ability to concentrate nanoparticles at the tumor site resulted in a significantly greater reduction in tumor volume compared to tumors treated with a 20-fold higher dose of the free drug.

Tau peptides and tau mutant protein aggregation inhibition by cationic polyethyleneimine and polyarginine.

Tau protein plays a major role in Alzheimer's disease. The tau protein loses its functionality by self-aggregation due to the two six-amino acid sequences VQIVYK and VQIINK of the protein. Hence it is imperative to find therapeutics that could inhibit the self-aggregation of this tau peptide fragments. Here, we study the inhibitory potential of a cationic polymer polyethyleneimine (PEI) and a cationic polypeptide arginine (Arg) on the aggregation of VQIVYK, and GKVQIINKLDL peptides, and tau mutant protein (P301L), found frequently in taupathy. Various characterization methods are employed including thioflavin S, transmission electron microscopy, and dynamic light scattering to study the aggregation/inhibition process in vitro. Results show that PEI and Arg significantly inhibit tau peptides and protein aggregation. The study could be applied to understand tau protein aggregation mechanism in the presence of cationic polymers.

Polyarginine and its analogues inhibit p53 mutant aggregation and cancer cell proliferation in vitro.

Arginine, a cationic amino acid is known to stabilize proteins under harsh conditions. It is widely used to stabilize protein aggregation, and to correct protein folding during protein production. Hence it would be a good therapeutic candidate for treating protein aggregation related diseases. Recent reports suggest, that the aggregation of tumor suppressor protein p53 is one of the leading causes of tumor progression. When mutated, p53 protein aggregates, loses its function leading to unwanted cell growth and ultimately results in tumor. Here in this study we focus on the inhibitory effects of polyarginine and its analogues polyornithine, canavanine, and citrulline on the inhibition of p53 mutant peptide aggregation, and p53 mutant cancer cell proliferation inhibition in vitro. Biochemical assays and cell toxicity studies were used to characterize the study. The results show that polyarginine, and polyornithine, in micromolar concentrations, significantly inhibits p53 conserved peptide aggregation, and the cell proliferation of p53 mutant cancer cells. Hence they could be promising candidates for treating p53 mutant/misfolded protein aggregation associated cancer.

Inhibition of p53 Mutant Peptide Aggregation In Vitro by Cationic Osmolyte Acetylcholine Chloride.

Mutations of tumor suppressor protein p53 are present in almost about 50% of all cancers. It has been reported that the p53 mutations cause aggregation and subsequent loss of p53 function, leading to cancer progression. Here in this study we focus on the inhibitory effects of cationic osmolyte molecules acetylcholine chloride, and choline on an aggregation prone 10 amino acid p53 mutant peptide WRPILTIITL, and the corresponding wildtype peptide RRPILTIITL in vitro. The characterization tools used for this study include Thioflavin- T (ThT) induced fluorescence, transmission electron microscopy (TEM), congo red binding, turbidity, dynamic light scattering (DLS), and cell viability assays. The results show that acetylcholine chloride in micromolar concentrations significantly inhibit p53 mutant peptide aggregation in vitro, and could be promising candidate for p53 mutant/ misfolded protein aggregation inhibition.

Aqueous Two Phase System Assisted Self-Assembled PLGA Microparticles.

Here, we produce poly(lactide-co-glycolide) (PLGA) based microparticles with varying morphologies, and temperature responsive properties utilizing a Pluronic F127/dextran aqueous two-phase system (ATPS) assisted self-assembly. The PLGA polymer, when emulsified in Pluronic F127/dextran ATPS, forms unique microparticle structures due to ATPS guided-self assembly. Depending on the PLGA concentration, the particles either formed a core-shell or a composite microparticle structure. The microparticles facilitate the simultaneous incorporation of both hydrophobic and hydrophilic molecules, due to their amphiphilic macromolecule composition. Further, due to the lower critical solution temperature (LCST) properties of Pluronic F127, the particles exhibit temperature responsiveness. The ATPS based microparticle formation demonstrated in this study, serves as a novel platform for PLGA/polymer based tunable micro/nano particle and polymersome development. The unique properties may be useful in applications such as theranostics, synthesis of complex structure particles, bioreaction/mineralization at the two-phase interface, and bioseparations.

Shear-Activated Nanoparticle Aggregates Combined With Temporary Endovascular Bypass to Treat Large Vessel Occlusion.

BACKGROUND AND PURPOSE: The goal of this study is to combine temporary endovascular bypass (TEB) with a novel shear-activated nanotherapeutic (SA-NT) that releases recombinant tissue-type plasminogen activator (r-tPA) when exposed to high levels of hemodynamic stress and to determine if this approach can be used to concentrate r-tPA at occlusion sites based on high shear stresses created by stent placement. METHODS: A rabbit model of carotid vessel occlusion was used to test the hypothesis that SA-NT treatment coupled with TEB provides high recanalization rates while reducing vascular injury. We evaluated angiographic recanalization with TEB alone, intra-arterial delivery of soluble r-tPA alone, or TEB combined with 2 doses of intra-arterial infusion of either the SA-NT or soluble r-tPA. Vascular injury was compared against stent-retriever thrombectomy. RESULTS: Shear-targeted delivery of r-tPA using the SA-NT resulted in the highest rate of complete recanalization when compared with controls (P=0.0011). SA-NT (20 mg) had a higher likelihood of obtaining complete recanalization as compared with TEB alone (odds ratio 65.019, 95% confidence interval 1.77, >1000; P=0.0231), intra-arterial r-tPA alone (odds ratio 65.019, 95% confidence interval 1.77, >1000; P=0.0231), or TEB with soluble r-tPA (2 mg; odds ratio 18.78, 95% confidence interval 1.28, 275.05; P=0.0322). Histological analysis showed circumferential loss of endothelium restricted to the area where the TEB was deployed; however, there was significantly less vascular injury using a TEB as compared with stent-retriever procedure (odds ratio 12.97, 95% confidence interval 8.01, 21.02; P<0.0001). CONCLUSIONS: A novel intra-arterial, nanoparticle-based thrombolytic therapy combined with TEB achieves high rates of complete recanalization. Moreover, this approach reduces vascular trauma as compared with stent-retriever thrombectomy.

Nanoparticle targeting of anti-cancer drugs that alter intracellular signaling or influence the tumor microenvironment.

Nanoparticle-based therapeutics are poised to become a leading delivery strategy for cancer treatment because they potentially offer higher selectivity, reduced toxicity, longer clearance times, and increased efficacy compared to conventional systemic therapeutic approaches. This article reviews existing nanoparticle technologies and methods that are used to target drugs to treat cancer by altering signal transduction or modulating the tumor microenvironment. We also consider the implications of recent advances in the nanotherapeutics field for the future of cancer therapy.

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics.

Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-α. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.

Shear-activated nanotherapeutics for drug targeting to obstructed blood vessels.

Obstruction of critical blood vessels due to thrombosis or embolism is a leading cause of death worldwide. Here, we describe a biomimetic strategy that uses high shear stress caused by vascular narrowing as a targeting mechanism--in the same way platelets do--to deliver drugs to obstructed blood vessels. Microscale aggregates of nanoparticles were fabricated to break up into nanoscale components when exposed to abnormally high fluid shear stress. When coated with tissue plasminogen activator and administered intravenously in mice, these shear-activated nanotherapeutics induce rapid clot dissolution in a mesenteric injury model, restore normal flow dynamics, and increase survival in an otherwise fatal mouse pulmonary embolism model. This biophysical strategy for drug targeting, which lowers required doses and minimizes side effects while maximizing drug efficacy, offers a potential new approach for treatment of life-threatening diseases that result from acute vascular occlusion.

Inhibition of mammary tumor growth using lysyl oxidase-targeting nanoparticles to modify extracellular matrix.

A cancer nanotherapeutic has been developed that targets the extracellular matrix (ECM)-modifying enzyme lysyl oxidase (LOX) and alters the ECM structure. Poly(d,l-lactide-co-glycolide) nanoparticles (∼220 nm) coated with a LOX inhibitory antibody bind to ECM and suppress mammary cancer cell growth and invasion in vitro as well as tumor expansion in vivo, with greater efficiency than soluble anti-LOX antibody. This nanomaterials approach opens a new path for treating cancer with higher efficacy and decreased side effects.

A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells.

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 µm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.

Polymeric nanomaterials for islet targeting and immunotherapeutic delivery.

Here we report a proof-of-concept for development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune type 1 diabetes. Modified with a unique islet-homing peptide, these polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 200-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. Our findings also underscore the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression.