Nanoscale flow cytometry to distinguish subpopulations of prostate extracellular vesicles in patient plasma.

OBJECTIVE: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance. MATERIALS AND METHODS: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA). RESULTS: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer. CONCLUSION: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate.

Establishing the effects of mesoporous silica nanoparticle properties on in vivo disposition using imaging-based pharmacokinetics.

The progress of nanoparticle (NP)-based drug delivery has been hindered by an inability to establish structure-activity relationships in vivo. Here, using stable, monosized, radiolabeled, mesoporous silica nanoparticles (MSNs), we apply an integrated SPECT/CT imaging and mathematical modeling approach to understand the combined effects of MSN size, surface chemistry and routes of administration on biodistribution and clearance kinetics in healthy rats. We show that increased particle size from ~32- to ~142-nm results in a monotonic decrease in systemic bioavailability, irrespective of route of administration, with corresponding accumulation in liver and spleen. Cationic MSNs with surface exposed amines (PEI) have reduced circulation, compared to MSNs of identical size and charge but with shielded amines (QA), due to rapid sequestration into liver and spleen. However, QA show greater total excretion than PEI and their size-matched neutral counterparts (TMS). Overall, we provide important predictive functional correlations to support the rational design of nanomedicines.

Understanding the Connection between Nanoparticle Uptake and Cancer Treatment Efficacy using Mathematical Modeling.

Nanoparticles have shown great promise in improving cancer treatment efficacy while reducing toxicity and treatment side effects. Predicting the treatment outcome for nanoparticle systems by measuring nanoparticle biodistribution has been challenging due to the commonly unmatched, heterogeneous distribution of nanoparticles relative to free drug distribution. We here present a proof-of-concept study that uses mathematical modeling together with experimentation to address this challenge. Individual mice with 4T1 breast cancer were treated with either nanoparticle-delivered or free doxorubicin, with results demonstrating improved cancer kill efficacy of doxorubicin loaded nanoparticles in comparison to free doxorubicin. We then developed a mathematical theory to render model predictions from measured nanoparticle biodistribution, as determined using graphite furnace atomic absorption. Model analysis finds that treatment efficacy increased exponentially with increased nanoparticle accumulation within the tumor, emphasizing the significance of developing new ways to optimize the delivery efficiency of nanoparticles to the tumor microenvironment.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

BACKGROUND: The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. METHODS: We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. RESULTS: To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. CONCLUSIONS: While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells.

BACKGROUND: Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. NEW METHOD: We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. RESULTS: CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). COMPARISON WITH EXISTING METHOD(S): Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. CONCLUSIONS: CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases.

Mesoporous Silica Nanoparticle-Supported Lipid Bilayers (Protocells) for Active Targeting and Delivery to Individual Leukemia Cells.

Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry.

BACKGROUND: Extracellular vesicles released by prostate cancer present in seminal fluid, urine, and blood may represent a non-invasive means to identify and prioritize patients with intermediate risk and high risk of prostate cancer. We hypothesize that enumeration of circulating prostate microparticles (PMPs), a type of extracellular vesicle (EV), can identify patients with Gleason Score≥4+4 prostate cancer (PCa) in a manner independent of PSA. PATIENTS AND METHODS: Plasmas from healthy volunteers, benign prostatic hyperplasia patients, and PCa patients with various Gleason score patterns were analyzed for PMPs. We used nanoscale flow cytometry to enumerate PMPs which were defined as submicron events (100-1000nm) immunoreactive to anti-PSMA mAb when compared to isotype control labeled samples. Levels of PMPs (counts/µL of plasma) were also compared to CellSearch CTC Subclasses in various PCa metastatic disease subtypes (treatment naïve, castration resistant prostate cancer) and in serially collected plasma sets from patients undergoing radical prostatectomy. RESULTS: PMP levels in plasma as enumerated by nanoscale flow cytometry are effective in distinguishing PCa patients with Gleason Score≥8 disease, a high-risk prognostic factor, from patients with Gleason Score≤7 PCa, which carries an intermediate risk of PCa recurrence. PMP levels were independent of PSA and significantly decreased after surgical resection of the prostate, demonstrating its prognostic potential for clinical follow-up. CTC subclasses did not decrease after prostatectomy and were not effective in distinguishing localized PCa patients from metastatic PCa patients. CONCLUSIONS: PMP enumeration was able to identify patients with Gleason Score ≥8 PCa but not patients with Gleason Score 4+3 PCa, but offers greater confidence than CTC counts in identifying patients with metastatic prostate cancer. CTC Subclass analysis was also not effective for post-prostatectomy follow up and for distinguishing metastatic PCa and localized PCa patients. Nanoscale flow cytometry of PMPs presents an emerging biomarker platform for various stages of prostate cancer.

Protocells: Modular Mesoporous Silica Nanoparticle-Supported Lipid Bilayers for Drug Delivery.

Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on.

Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles.

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.

Expression profiles of androgen independent bone metastatic prostate cancer cells indicate up-regulation of the putative serine-threonine kinase GS3955.

PURPOSE: We established gene expression profiles by gene array analysis in the LNCaP model of human prostate cancer progression and evaluated genes differentially expressed in the androgen independent and bone metastatic C4-2 cell line compared to the androgen dependent and nonmetastatic parental LNCaP cell line. MATERIALS AND METHODS: Gene expression profiles were generated using Atlas cDNA arrays (Clontech, Palo Alto, California), comprising 1,176 genes. Intrinsic expression of the novel serine/threonine kinase GS3955 in LNCaP, C4-2 and PC-3 prostate cancer cells, and expression when stimulated with growth factors, was monitored by real-time reverse transcriptase-polymerase chain reaction. Furthermore, expression in human tumor specimens was evaluated. Cellular localization of GS3955 protein was analyzed by expressing it as a fusion with green fluorescent protein. RESULTS: Comparable numbers of genes were up-regulated and down-regulated in C4-2 compared to LNCaP. The novel serine/threonine kinase GS3955 was markedly up-regulated (greater than 40-fold) in C4-2, differentially regulated in LNCaP and C4-2 by insulin-like growth factor-1, and variably expressed in human prostate tumor specimens. Moreover, GS3955 was shown to localize in the cell cytoplasm and nucleus. CONCLUSIONS: Differential expression and mitogenic regulation of the serine/threonine kinase GS3955 in LNCaP and C4-2 suggest its functional involvement in the development of androgen independence and/or metastatic potential. GS3955 is also expressed in human prostate cancer specimens and further analysis may provide insights into the biology of prostate cancer progression.