December 1-6, 2013 | BostonMeeting Chairs: Charles Black, Elisabetta Comini, Gitti Frey, Kristi Kiick, Loucas Tsakalakos
Cisplatin and other DNA-damaging chemotherapeutics are widely used to treat a broad spectrum of malignancies. However, their application is limited by the emergence of tumor chemoresistance. Most mutations that result from DNA damage are the consequence of error-prone translesion DNA synthesis (TLS), which could thus be responsible for the acquired resistance against DNA-damaging agents. Recent studies have shown that the suppression of crucial gene products (e.g. REV1, REV3L) involved in the error-prone TLS pathway reduces the frequency of acquired drug resistance of relapsed tumors so that they remain susceptible to subsequent chemotherapy. In this context, combining conventional DNA-damaging chemotherapy with small interfering RNA (siRNA)-based therapeutics represents a promising strategy for treating patients with malignances. Towards this end, we have developed a versatile nanoparticle (NP) platform to simultaneously deliver a cisplatin prodrug and REV1/REV3L specific siRNAs to the same tumor cells. NPs are formulated through self-assembly of a biodegradable poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) diblock copolymer and a self-synthesized cationic lipid. We demonstrated the potency of the siRNA-containing NPs to efficiently knockdown target genes both in vitro and in vivo. The therapeutic efficacy of NPs containing both cisplatin prodrug and REV1/REV3L specific siRNAs was further investigated in vitro and in vivo. qRT-PCR results showed that the NPs exhibited a significant and sustained suppression of both genes in tumors for up to 3 days after a single dose. Administering these NPs revealed a synergistic effect on tumor inhibition in an LNCaP xenograft mouse model that was strikingly more effective than platinum monotherapy.
Introduction: Layer-by-Layer (LbL) assembly is a highly tunable, modular approach to surface-limited functionalization of materials with nanoscale precision over the composition and properties of the film components. This high level of control affords the capability to design systems that are multi-functional in nature, with temporal and spatial control over the release of a diverse range of materials and therapeutics of interest. Application of this approach to nanomedicine has been successful in designing hydrated, protein-resistive long-circulating nanoparticles, as well as systems that shed a hydrated shell used for enhanced persistence in the bloodstream and EPR-based accumulation in the hypoxic tumor microenvironment, whereby exposure of a positively-charged material facilitates rapid uptake by tumor cells. This has driven much interest in further characterizing these systems as drug carriers, with the means to sustain drug in complex biological settings, such as the bloodstream. Materials and Methods: Solid nanoparticles (PLGA, quantum dots) provide the foundation for LbL assembly, which is conducted via incubation of particle systems in excess polyelectrolyte materials (synthetic polypeptides, such as poly(L-lysine); glycosoaminoglycans, such as hyaluronic acid, alginate) at physiologic pH for iterative adsorption of materials on the basis of electrostatics. Encapsulating a model drug, near-IR emitting cardiogreen, in a complementary near-IR labeled LbL nanoparticle, real-time dual-tracking capabilities in vivo provided exquisite temporal and spatial resolution over the biological performance of systems generated. Multiple readouts were generated to evaluate these systems on the basis of fluorescence recovery (biodistribution, blood circulation, feces drug recovery). This approach is also being adapted to systems investigating molecular targeting in vivo, as well as multi-drug loaded nanoparticles for synergistic treatment of cancer. Results and Discussion: Using a two-color imaging approach and IVIS whole-animal fluorescence imaging, multiple readouts regarding the stability and drug-retention capabilities of a series of LbL nanoparticles were generated. Variation on number of film bilayers and terminal layer properties were evaluated as a means of understanding the biological performance of these systems. It was found that highly hydrated, protein-resistive anionic terminal coatings, such as hyaluronic acid and alginate, significantly improve the pharmacokinetics of the drug and particle core system being used as a template for LbL. This has facilitated on-going work evaluating LbL as a means to molecularly engage cell receptors in vivo for targeted delivery, as well as programmable release of film components for staged delivery of synergistic combinations of drugs for treatment of invasive cancer types, such as triple negative breast cancer, where timing has been shown to enhance drug combination efficacy.
The interaction of living cells with micro or nanoparticles has become more important as particles are increasingly used for drug delivery and other biomedical applications. Shape, surface chemistry and size characteristics can be designed to increase drug delivery efficiency by controlling the targeting and clearance of the synthetic particles. In particular, it has been found that the local shape of an anisotropic particle in contact with the cell determines the internalization rate of the particle. This insight drives the desire to design anisotropic particles that orient themselves on the cell surface to either promote or resist internalization depending on the desired purpose. One can imagine using the controlled interactions of anisotropic particles to form stable cell-biomaterial hybrids for drug delivery. Inspired by the use of chemically non-uniform Janus or patchy particles to control the local orientation of synthetic particles in colloid systems, we designed a tube-shaped, chemically non-uniform microparticle with the capability to control its orientation on cell surfaces. These anisotropic microtubes were fabricated using a sacrificial membrane template method and were designed to present cell-adhesive ligands on the ends of the tubes and a cell-resistant surface on the sides. In this work the cell-adhesive region incorporated hyaluronic acid to interact with the CD44 receptor on the surface of lymphocyte B-cells and a highly swollen polyelectrolyte multilayer was chosen to resist cell adhesion. Our results show that by altering the presentation of polymer on the end versus the side we can alter the proportion of cells connecting to the end of tubes versus the side of tubes. This simple method to make anisotropic and chemically non-uniform particles is generalizable and could incorporate a wide variety of materials for potential applications in stimuli responsive presentation of cell-adhesive regions or drug release. Recent literature also has shown that the conjugation of material to the cell surface is useful for cell-mediated drug delivery and by controlling the orientation of the material on the cell surface we can better design the cellular response to the synthetic materials. This advancement opens the possibility to design new cell-biomaterial hybrids for a variety of biomedical applications.
With concerns existing in conventional chemotherapeutic regimens such as low drug efficacy and severe systemic toxicity, the development of novel drug delivery systems (DDS) has been highly required. Nanotechnology has been employed to address the current challenges while formulating drug delivery carriers with an improvement of therapeutic efficiency by an increase of delivered drug as well as reduction of dose-limiting toxicity. Liposome is one of the nano-sized drug carriers that have been approved in clinical use. There are liposomal DDS products that have been already commercialized in tumor therapeutics, which mainly rely on the ability of passive tumor targeting via enhanced permeability and retention (EPR) effect resulting from the leaky tumor vasculature. However, a slow sustained release of drug from cumulated liposomes has not led to an impressive increase of anti-tumor efficacy. Thermo-sensitive liposomes (TSLs) have shown the ability to trigger encapsulated drugs by locally treated thermal stimuli within the region of tumor, resulting in an abrupt exposure of highly concentrated drugs to tumor tissues while TSLs pass through tumor vessels. Celsion, one of the leading groups developing TSLs for tumor therapeutics, had entered phase III of clinical trials with lysolipid-containing thermo-sensitive liposome (LTSL); however, they have ended up not meeting the primary endpoint in the clinical study. Some other studies claimed that a limitation of LTSL is possibly from a short half life and premature leakage of drugs from liposomes that led to low accumulation of bioavailable drugs. In contrast, TSL with improved stability in plasma resulted in enhanced anti-tumor efficacy. As interests upon TSL have increased along with ongoing clinical trials, there are few protocols setup for the use of TSL to treat tumors that have been established through the process optimization with a desirable heat source.We have previously developed short chain elastin-like polypeptide incorporating thermo-sensitive liposome (STL) that has 4-times better stability than that of LTSL with high sensitivity upon thermal stimulation at the range of mild hyperthermia (42°C). In this study, we examined various approaches for the use of STL in tumor therapy. First, we optimized the protocols to obtain the largest amount of drug accumulation; namely, conditions for mild hyperthermia treatment to enhance the EPR effect, and time course analysis with lag-time after injecting STL to be accumulated in tumor tissues during systemic circulation. Second, we examined the timings of heat stimuli at tumor sites for drug-bursting while or after STLs are accumulated in tumor tissues. We also compared the efficiency of two different heat sources, a warm water bath (42°C) and high intensity focused ultrasound (HIFU) in the use of STL.
Despite recent improvements in cancer therapy, intraperitoneal (IP) carcinomatosis of pancreatic ductal adenocarcinoma origin remains a significant and unresolved therapeutic challenge with 5 year survival rates of ~5%. Treatment failure is attributed, in part, to the inability to detect and remove microscopic disease. To address this problem, hyperthermic IP chemotherapy (e.g. paclitaxel (Pax)) has been used and shown to provide a modest benefit in some peritoneal cancers (e.g. ovarian). However, toxicity of the carrier, a 50/50 mixture of Cremophor/ethanol (C/E), and rapid clearance of IP administered Pax has prevented this treatment from being curative, with patients ultimately relapsing due to residual disease. In particular, 50% of the injected Pax is cleared within 3 hours even as systemic side effects persist. Since Pax only acts during cell replication and, at any given time, only 10-15% of tumor cells are expected to be in mitosis, tumoral response to Pax is reduced due to short exposure times. In order to prolong the delivery of Pax to tumor tissues, we have designed a polymeric nanoparticle drug delivery system. Nanoparticles are frequently leveraged as drug delivery systems for their ability to encapsulate high concentrations of insoluble or readily degraded chemotherapeutic agents directly to tumor sites while minimizing systemic toxicity and adverse side effects. We have developed polymeric pH-responsive expansile nanoparticles (eNPs) that achieve intracellular delivery of hydrophobic drugs (e.g. Pax) via pH-triggered swelling in the late endosome. Acetal-protecting groups within the polymer are cleaved at mildly acidic pH resulting in a compositional change within the eNP from hydrophobic to hydrophilic resulting in infiltration of water into the nanoparticle. Hydration of eNPs, and subsequent swelling, allows Pax to diffuse out leading to intracellular delivery/accumulation of Pax and, consequently, cell death. To investigate the efficacy of Pax-loaded eNPs (Pax-eNPs), we used a human pancreatic cancer line (Panc-1) to develop cancer stem-like cells (CSCs), which more accurately mimic the increased tumorigenesis, chemotherapy- and anoikis-resistance seen clinically. Results demonstrate rapid internalization (characterized via confocal microscopy and flow cytometry) of fluorescent-rhodamine-labeled eNPs (Rho-eNPs) within a Panc-1-CSCs as well as dose-dependent cytotoxicity of Pax-eNPs against this same line. Furthermore, in vivo results demonstrate that Rho-eNPs administered intraperitoneally localize to both micro- and macroscopic tumors without the need for targeting ligands. These results suggest that eNPs may provide a useful means of delivering high doses of Pax to peritoneal carcinomatoses of pancreatic ductal carcinoma origin, thereby improving patient outcomes.
Human glioblastoma multiforme (hGBM) is a common and aggressive form of brain tumor. Biomaterials able to replicate elements of the native tumor microenvironment are a critical topic in the field of cancer research. Such a system could serve as a diagnostic platform for clinical assessment of therapeutic strategies. We have developed an adaptable hydrogel system based on methacrylated gelatin (GelMA) backbone which allows for systematic alteration of the adhesive and biophysical environment. Using this tool we have begun to explore the impact of the local matrix microenvironment on glioma malignancy using the U87MG glioma cell line. Critically, grafting brain-mimetic hyaluronic acid (HA) into the hydrogel network was found to induce significant, dose-dependent alterations of markers of glioma malignancy versus non-grafted 3D gelatin or PEG hydrogels. Clustering of glioma cells was observed exclusively in HA containing gels and expression profiles of malignancy-associated genes were found to vary biphasically with incorporated HA content. We also found HA-induced expression of MMP-2 was blocked by +EGFR signaling (a common mutation found in highly-malignant GBM tumors), suggesting a connection between CD44 and EGFR in glioma malignancy. We have now combined the GelMA hydrogel system with a microfluidic mixer to create overlapping mixtures of the multiple hydrogel precursor suspensions prior to UV-mediated photopolymerization. Using this approach we create single hydrogels containing coincident patterns of glioma-inspired cell, matrix, and biomolecular cues. We can subsequently image individual cells or cell masses via confocal microscopy as well as remove discrete regions for subsequent genomic and bioactivity analyses. Using this tool we have examined the impact of transitions across gradient hydrogels that mimic those seen across the heterogeneous glioma tumor mass (e.g., glioma core vs. periphery). Notably, gradients in matrix crosslinking density and HA incorporation induced changes in U87MG cell morphology, gene expression, and malignant phenotype as seen in disparate monolithic hydrogels. Moving forwards, we are using this tool as a brain tumor biochip to examine the impact of increasingly heterogeneous environments (incorporating additional cellular and biomolecular cues) on glioma cell malignancy with the goal of identifying linkages between biophysical environments and therapeutic efficacies.
Hybrid plasmonic-superparamagnetic agglomerates [1,2] (less than 100 nm in diameter) consisting of multiple SiO2 -coated Au and Fe2O3 nanoparticles (~30 nm in diameter each) are made reproducibly by scalable gas-phase flame aerosol technology. By finely tuning the Au interparticle distance by the SiO2 film thickness  (or content), the plasmonic coupling of gold nanoagglomerates is closely controlled along with the optical absorption to the near-IR spectral region. The SiO2 shell facilitates dispersion and prevents the reshaping or coalescence of Au particles during laser irradiation facilitating their use multiple treatments. The effectiveness of such plasmonic nanostructures as photothermal agents is demonstrated on human breast cancer cells by near-IR laser irradiation at low power (4.9 W/cm2) and 785 nm for 4 minutes.References Sotiriou G. A., Hirt A. M., Lozach P. Y., Teleki A., Krumeich F. and Pratsinis S. E. Hybrid, silica-coated, Janus-like plasmonic-magnetic nanoparticles. Chem. Mater.23, 1985-1992 (2011). G.A. Sotiriou, Biomedical Applications of Multifunctional Plasmonic Nanoparticles. WIREs Nanomed. Nanobiotechnol.5, 19-30 (2013). Sotiriou G. A., Sannomiya T., Teleki A., Krumeich F., Vörös J. and Pratsinis S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors. Adv. Funct. Mater.20, 4250-4257 (2010).
Graphene oxide (GO) has been widely investigated for biomedical applications due to its unique physical, mechanical and optical properties. In particular, GO and reduced GO have a high photothermal effect under NIR irradiation due to their effective light-to-heat conversion compared to other carbon allotropes. Here, we report transdermal nano-sized GO (NGO) - hyaluronate (HA) conjugates for photo-ablation therapy of melanoma skin cancer using NIR laser. Melanoma is less common, but it is one of the most dangerous skin cancers and the main cause of skin cancer related death. Melanoma can infiltrate and spread deeply into the skin. To our knowledge, this is the first report to deliver NGO through transdermal pathway and treat skin cancer with NIR irradiation. The NGO-HA appeared to be transdermally delivered to the skin cancer in mice with highly expressed HA receptors and a relatively leaky structure rendering the enhanced permeation of nanoparticles. After bioimaging for the transdermal delivery of NGO-HA labeled with NIR fluorescent Hilyte 647 dye, we successfully demonstrated the photo-ablation therapy of melanoma skin cancer in mice. The anti-tumor photo-ablation effect was confirmed by ELISA for caspase-3 activity, histological analysis and immuno-histochemical TUNEL assay. Minimizing the possible side effect of NGO in the body, this system is likely to be much safer than systemic delivery systems, because NGO-HA is directly and locally accumulated in tumor tissues by the transdermal pathway. In combination with drug loading to NGO-HA by - stacking, this system can be applied for transdermal chemo- and photothermal therapy of various skin cancers.
Extraction of intracellular signal molecules is essential for interrogation of cellular pathways and characteristics. However, the extraction in live cells still remains as a significant challenge. Here our experimental results show that nanospearing could serve as a promising technique for cell interrogation. The basic idea is to conduct external magnetic field-assisted driving of magnetic nanotubes (MNTs) to penetrate cell body and carry out the molecules. The MNTs are synthesized through poly-carbonate template-assisted electrochemical deposition. Electropolymerization of a protective layer of biomaterials was used to render the surface of MNTs to be biocompatible. Under magnetic field-driving, MNTs penetrated through the cells without causing cell death. Green fluorescent protein (GFP) expressed from GFP-plasmid was used to visualize the process. During the progress of penetration, some GFP molecules entrapped in the tubes were forced out of the cells by external magnetic field. Furthermore, real-time polymerase chain reaction analysis detected β-actin mRNA and DNA in the penetrated-MNTs. These results implicate that nanospearing can extract cellular signal molecules from live cells. It could lay out a novel approach to investigate cellular pathways of pathogenesis and help to explore novel diagnosis and therapeutics of diseases.
There has been a growing interest in using nanoparticles as drug delivery vehicles, especially for cancer treatment [1, 2]. Nanoparticles of different sizes and shapes have been produced from a variety of materials and the surface chemistry of these nanoparticles can be further tailored to evade the immune system and/or facilitate their selective attachment at the targeted sites. However, little is known about the flow dynamics, or rheology, of nanoparticles in blood flow, which must be understood if the nanoparticles are to be administered intravenously. Further, Decuzzi et al.  proposed theoretically that the interactions between the blood vessel walls and nanoparticles can lead to a “margination” phenomenon wherein the nanoparticles trend toward the periphery of blood vessels. The implication is a higher chance for the nanoparticles to diffuse into the tumor through the leaky vasculature typically found near tumor sites. To advance our fundamental understanding of margination, we have constructed microfluidic devices that mimic blood vessel bifurcation. Fluorescently tagged polystyrene nanoparticles of varying size and shape have been used as a model system. The trajectory of these nanoparticles has been characterized to explore the effects of flow geometry, particle size and shape, and suspending medium rheology on the margination propensity. The findings may have far-reaching implications on the rational design of nanoparticles to allow more specific delivery of anticancer drug into tumors. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1247393 and by the National Science Foundation under Grant No. 1250661. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation. This research is also supported by the Department of Defense Mentor-Predoctoral Fellow Research Award program under award number W81XWH-10-1-0434. Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense. References1. Ferrari, M., Cancer nanotechnology: opportunities and challenges. Nat. Rev. Cancer, 5(3): 161-171 (2005).2. Davis, M.E., Fighting cancer with nanoparticle medicines - The nanoscale matters. MRS Bulletin, 37: 828-835 (2012).3. Decuzzi, P., Lee. S., Bushan, B., Ferrari, M., A theoretical model for the margination of particles within blood vessels. Annals Biomed. Eng., 33(2): 179-190 (2005).
The use of particles as delivery vehicles is considered a powerful tool in the fields of biomedical imaging and drug delivery. Recently, particle shape has been recognized as an important factor in biological processes affecting particle cellular uptake and vascular dynamics. We demonstrate a simple approach to fabricate biocompatible monodisperse hollow microparticles of controlled geometry. The robust hemispherical, spherical and cubical particles are obtained by drying multilayer capsules of hydrogen-bonded poly(N-vinylpyrrolidone)/tannic acid, (PVPON/TA)n. Drying spherical capsules results in hemispherical particles if 15
Metastatic and malignant melanoma remains highly lethal cancer. BNCT (boron neutron capture therapy) is single cell-selective radiation therapy for cancer. So, BNCT has been attracted great deal of attention as a potent modality for malignant melanoma. The success of BNCT depends on the boron delivery system to accumulate effectively and deeply inside the tumor cells. Clinically, Boronophenylalanine (BPA) and Sodium borocaptate (BSH) are currently used for BNCT as boron carriers. However, these compounds have some disadvantages on accumulation, water-solubility, or selectivity toward tumor tissue. On the other hand, it has been well known that kojic acid possesses a whitening ability to melanocytes by a strong tyrosinase inhibition. This fact suggests that kojic acid could work as effective ligand for melanoma-targeting. In order to construct a novel boron delivery system for melanoma-targeting BNCT, we used carborane-kojic acid conjugate (CKA). Because CKA shows little water-solubility, various cyclodexitrins were used as a solubilizer. As the inclusion complex of hydroxypropyl-β-cyclodextrin (HP-β-CD) provides the highest concentration of CKA solution, herein, the CKA/ HP-β-CD complex was estimated as boron carrier for melanoma-targeting BNCT. Water-soluble CKA complexes were effectively prepared with HP-β-CD by using mixing with vortex-mixer. After addition of CKA/HP-β-CD to culture medium of B16BL6 (murine melanoma) and colon26 (murine colorectal cancer), relative cell viability was estimated in 24 hours. CKA/HP-β-CD shows little toxicity under 40 ppmB. Therefore, Cellular uptake and cellular distribution of CKA/HP-β-CD were evaluated within 10 ppmB. CKA/HP-β-CD was taken up more efficiently by B16BL6 than colon26. Uptake by B16BL6 was inhibited with excess kojic acid/HP-β-CD complex. These results indicate CKA/HP-β-CD possesses melanoma affinity and selectivity. Moreover, CKA/HP-β-CD was localized at nucleus in 1 hour after treatment. Therefore, CKA/HP-β-CD might perform BNCT effectively by its nuclear accumulation. The therapeutic and antitumor efficiency of CKA/HP-β-CD were evaluated by using tumor-bearing mice implanted with B16BL6 cells. CKA/HP-β-CD and L-BPA fructose complex were injected by i.p before 1 hr of irradiation. Neutron irradiation was carried out at Kyoto University Research Reactor (5 MW, 18 min, 5.0×1012 neutron/cm2). By irradiation, proliferation and antitumor efficiency of BNCT were improved within concentration-dependent and neutron fluence-dependent. Moreover, CKA/HP-β-CD shows similar or superior tumor suppression effect to L-BPA.In summary, CKA/HP-β-CD can deliver toward melanoma selectively and effectively. Therefore, CKA/HP-β-CD can improve the survival of the tumor-bearing mice as effective as L-BPA. This boron carrier is promising for melanoma BNCT.
Stimuli-sensitive polymeric nanoparticles have emerged as a promising carrier for triggered release of hydrophobic anticancer drugs. In this study, we synthesized a bioreducible amphiphilic diblock copolymer, composed of poly(ethyleneglycol) (PEG) and poly(γ-benzyl L-glutamate) (PBLG), bearing disulfide bond (PEG-SS-PBLG) as the potential carrier of doxorubicin (DOX). Owing to its amphiphilic nature, the amphiphilic copolymers formed nano-sized micelles (137nm in diameter) in aqueous conditions. The micelles were stable in the physiological condition (pH 7.4), whereas they were rapidly disassembled in the presence of glutathione (GSH), a thiol-containing tripeptide capable of reducing the disulfide bond. Doxorubicin (DOX), chosen as the model anticancer drug, was effectively encapsulated into the hydrophobic core of the micelle. At 10 mM GSH, DOX was completely released in 18 h from the micelles, whereas only 34% of DOX was released at 2mu;M GSH. Since GSH is abundant at the intracellular level, DOX-loaded PEG-SS-PBLG micelles exhibited higher toxicity to SCC7 cells than DOX-loaded PEG-b-PBLG micelles without the disulfide bond. These results suggest that the diblock copolymer bearing the bioreducible linker have potential as the carrier for the triggered intracellular drug delivery.
The development of biomolecules-nanoparticles conjugates is a topic of intense and growing interest for extending the applications of nanomaterials in biomedicine. Despite the recent advances, the biomedical applications of these materials are still limited, among other factors, by the low efficiency of functionalization, low stability and high toxicity. Overcoming these obstacles requires a complete understanding of the interactions between nanomaterials and biomolecules. Here, we present the development of jacalin-conjugated gold nanoparticles (AuNPs/jacalin) for leukemia cells detection with focus on the understanding and characterization of the nanoconjugates. Jacalin is a lectin that may specifically recognize a tumor-associated disaccharide that is overexpressed in most types of human cancers. AuNPs were synthesized in presence of poly(amido amine) generation 4 (PAMAM G4) and conjugated with a jacalin targeted with the fluorescein isothiocyanate (FITC). The AuNPs/jacalin formation is driven by an entropic process with good affinity, as revealed by isothermal titration calorimetry and quenching fluorescence measurements. Moreover, in vitro tests revealed that the AuNPs/jacalin-FITC complexes presented higher affinity against leukemia cells compared to normal ones. The nanoconjugates were successfully immobilized on specific electrodes for impedimetric detection of leukemia cells. Our findings are relevant for extending the understanding of the interactions between nanomaterials and biomolecules besides of their applications in biomedicine, especially for cancer cells detection.
Recent advances in nanomedicine have introduced a novel concept, theragnosis, to serve as a simultaneous therapeutic and diagnostic tool with the use of well-tailored nanoplatforms. This relatively new concept enables efficient delivery of therapeutic agent co-currently with real time monitoring of tumor region. Herein, we synthesized a novel gold protruding magnetic nanocomposite (GP-MNC) with the water soluble magnetic core as a magnetic resonance imaging agent and the gold protruded outer shell having optical resonance situated in near infrared (NIR) region for photothermal therapy. First, the carboxylate terminated MNCs with high saturation of magnetization were synthesized and used as a template for the seed mediated growth of gold spikes with the assistance of cetyltrimethylammonium bromide (CTAB). Increasing volumes of growth solution (2-20ml) were used for the formation of protrusions for tuning the surface plasmon resonance (SPR) in the NIR region for effective hyperthermia. CTAB layer was then replaced with monofunctional polyethylene glycol (CH3-PEG-SH) for in vivo stability of GP-MNCs. The morphology of the particles was studied by using High Resolution Transmission Electron Microscopy and the crystal structure was confirmed by X-ray diffraction. Surface chemical analysis of GP-MNCs was further evaluated by X-ray photoelectron spectroscopy. Biocompatiblity of the particles was affirmed by performing MTT assay for various GP-MNCs concentrations. To assess the potential of the as-synthesized particles for MRI guided photothermal therapy, Magnetic Resonance Imaging and in vitro/in vivo heat generation by laser irradiation against human gastric cancer cells (MKN-45 cells) were investigated. Accordingly, this study demonstrated that GP-MNCs are highly efficient MRI agents guiding effective hyperthermia effect in tumor cells, thus finding applications in clinical cancer therapies.
We have previously shown two different approaches for improving therapeutic delivery with mesoporous silica nanoparticles (MSNP): mechanized pore control and employing targeting ligands on the MSNPs. Pore control is achieved by functionalizing the silica surface with pH-sensitive nanovalves that utilize supramolecular chemistries to form host-guest complexes to block the pores. These nanovalves remain closed at physiological pH (7.4), protecting and trapping the cargo. When the MSNPs are endocytosed by cells and enter the lysosome (pH < 6), the pH-sensitive nanovalve will be activated— opening and releasing its contents to provide a stimulus-responsive and autonomous release of therapeutics. Alternatively, derivitizing the MSNP surface with biologically active molecules (e.g. folate, RGD, proteins, transferrin) causes increased cellular uptake. By integrating these two functions, a delivery system is fabricated that delivers therapeutics in a selective and controlled manner. In this work, the design and fabrication of an integrated, multifunctional mesoporous silica nanoparticle system, able to simultaneously image, target, and autonomously deliver therapeutics in vitro and in vivo is presented. The integrated, multimodal system is composed of targeting and fluorescent imaging modes, as well as pH-sensitive nanovalves that can autonomously activate and deliver cargo when exposed to pH lower than 6. Abiotic studies show the nanovalves&’ ability to function under the bulky protein and release cargo. In vitro studies demonstrated the improved delivery of doxorubicin into human pancreatic cancer cells (MiaPaCa-2) due to the efficacy of the targeting agent. Finally, the operation of this system in xenografts on SCID mice was demonstrated, proving that the system is capable of functioning in an in vivo model.
For the effective application of surface-enhanced Raman scattering (SERS) nanoprobes for in vivo targeting, the tissue transparency of the probe signals should be as high as it can be in order to increase detection sensitivity and signal reproducibility. Herein, we demonstrate near-infrared (NIR)-sensitive SERS nanoprobes (NIR SERS dots) for in vivo multiplex detection. NIR SERS dots consist of plasmonic Au/Ag hollow-shell (HS) assemblies on silica nanospheres and Raman labels. We modulate the optical properties of the HS assemblies by adjusting the HS nanostructures, which results in the red-shift of their extinction bands from 480 nm to 825 nm. The red-shifted plasmonic extinction of NIR SERS dots enables them to produce more enhanced SERS signals at NIR excitation window. Owing to the enhanced sensitivity of SERS in the NIR window, a single NIR SERS dot can produce very intense SERS signals with an average SERS enhancement factor value of 2.8×10^5. In addition, the NIR SERS dots exhibit very narrow distributions of SERS intensity with high reproducibility, which is very important for the quantitative detection of target molecules. The increased sensitivity of NIR SERS dot enables us to easily obtain SERS signals from deep tissues of up to 8-mm depth. Finally, the NIR SERS dots were successfully applied for in vivo multiplex detection by injecting them into the tissues of a live animal.
The siRNA delivery systems for cancer therapy have been primarily prepared by simple, ionic complexation between negatively charged siRNA and positively charged polymers. However, their poor stability and lack of targetability have been major hurdles for successful clinical translation. To overcome these limitations, we developed the hyaluronic acid-graft-poly(dimethylaminoethyl methacrylate) (HPD) conjugate which can form biostable complexes with siRNA via disulfide crosslinking. The in vitro gel-retardation images demonstrated that the disulfide-crosslinked HPD/siRNA complexes (C-siRNA-HPD) showed higher stability than that of uncrosslinked HPD/siRNA complexes (U-siRNA-HPD) in the presence of 50 % rat serum. Both of U-siRNA-HPD and C-siRNA-HPD were efficiently taken up by the cancer cells (B16F10) over-expressing CD44 via receptor-mediated endocytosis, whereas they were not significantly internalized by normal fibroblast cells (NIH3T3). When the RFP-expressing B16F10 cells were treated with C-siRNA-HPD, U-siRNA-HPD, and free siRNA, the highest gene-silencing effect was found for C-siRNA-HPD. From the non-invasive animal imaging results, it was found that C-siRNA-HPD complexes were significantly accumulated at the tumor site after systemic administration, resulting in remarkable decrease in RFP expression in vivo. Overall, these results suggested that the HPD conjugates are promising carriers of siRNA for tumor-targeted gene therapy.
Photodynamic therapy (PDT) is an emerging modality for the diagnosis and treatment of various cancers and metastases.1 Recently, research efforts have been devoted to the development of near-infrared photosensitizers for the treatment of deep-seated tumors.2,3 In this work, novel hydrophilic naphthalocyanines (Nc) were synthesized by metal-templated cyclization of substituted 2, 3-dicyanonaphthalene. These Ncs have strong absorbance at long wavelengths ( 770sim;780 nm); the light penetration through tissues in this region is approximately twice that of clinical used porphyrin-mediated PDT (630 nm). In addition, these Ncs can also efficiently generate singlet oxygen in DMF upon illumination with NIR light (lambda;= 750 ± 10 nm) with the Phi;Δ value as high as 0.66, which is the best singlet oxygen quantum yield that was reported for Nc so far, indicating these Ncs are superior NIR singlet oxygen generators. The in vitro photodynamic activities were investigated against Hela cells. In the absence of light, all these compounds were not cytotoxic. Upon irradiation with red light ( sim;1J/cm2), these Ncs were highly photocytotoxic with IC50 as low as 3.7 µM. Due to their complicated synthetic procedures and poor solubility in common organic solvents, few Ncs have been reported in the biomedical area so far despite their great potential in PDT. In particular, hydrophilic Ncs remain extremely rare. Our results indicate that these novel hydrophilic Ncs have efficient photodynamic activities in NIR region, and are therefore highly promising NIR photosensitizers for PDT of cancer.Reference1. Chem. Rev. 1999, 99, 2379.2. J. Am. Chem. Soc. 2008, 130, 15782.3. J. Am. Chem. Soc. 2010, 132, 7844.
Glioblastoma multiforme (GBM) is a high grade astrocytoma that exhibits extreme resistance to conventional therapies and commonly recurs following resection, leading to a dismal 12-month median survival post-diagnosis. However, most GBM tumors recur within 2 cm of the original tumor site, while extracranial metastasis is extremely rare. Therefore, a highly specific and efficacious treatment that can be administered locally after tumor resection could drastically improve patient outcomes by destroying remaining tumor cells, and thereby reducing GBM recurrence. Temozolomide (TMZ) is an alkylating agent that has been shown to demonstrate efficacy as a single agent for the treatment of recurrent GBM tumors. However, TMZ undergoes rapid hydrolysis under neutral and alkaline conditions and has a half-life of only 1.24 hours. Thus there is a need for a drug delivery system that can protect and deliver high local concentrations of pharmacologically active TMZ following tumor resection. We have engineered a nanoparticle (NP) drug delivery system to meet these criteria. NPs are a unique class of therapeutics that have gained momentum in recent years. Polymeric NPs have been especially used for the delivery of anti-cancer agents due to their ability to provide controlled and sustained drug release, as well as their ability to protect encapsulated agents from degradation while simultaneously minimizing systemic toxicity. We have engineered a novel, controlled, and pH-responsive expansile nanoparticle (eNP) system which can be used for the localized delivery of TMZ to GBM tumors. eNPs undergo a conformational change under mildly acidic conditions, such as those found in the endosome and lysosome, causing the particles to swell and release their payload intracellularly. Additionally, eNPs protect TMZ from degradation, and therefore demonstrate superior potency against a standard GBM cell line (U87), with an IC50 of ~200 ng/mL—a three order of magnitude improvement over free TMZ (IC50 of ~20,000 ng/mL). We herein demonstrate the internalization of rhodamine-labeled eNPs in both GBM cancer stem cells (CSCs) and “standard” tumor cells via flow cytometric analysis and confocal microscopy. We also show that TMZ-loaded eNPs demonstrate improved, dose-dependent cytotoxicity against both cell lines in vitro.
Calcium phosphate nanoparticles (CaPNs) have received increasing attention for biomedical applications due to its excellent biocompatibility and biodegradability. However, preparation of homogeneous and nano-sized CaPNs is still challenging due to irregular particle growth at the nano-level. In this study, linear polyethylenimne (LPEI) was coated onto the CaPNs for producing the well-controlled CaPNs, which was employed as a biocompatible and efficient carrier for the delivery of therapeutic microRNA, microRNA34a (miR34a). A miR34a, which acts as a tumor suppressor in many types of solid tumors, was chemically crosslinked to produce long chain miR34a (lc-miR34a). Naked miR34a and lc-miR34a were incorporated into the LPEI-CaPNs, respectively. The particle size of LPEI-CaPNs incorporating lc-miR34a was much smaller than that of LPEI-CaPNs incorporating naked miR34a. The LPEI-CaPNs incorporating lc-miR34a were successfully internalized into cytoplasm and suppressed the cell migration and proliferation for human prostate cancer cell, PC3, in contrast to the LPEI-CaPNs incorporating naked miR34a. The LPEI-CaPNs did not show any cytotoxicity. In conclusion, it was demonstrated that the LPEI-CaPNs with long chain microRNA might be an efficient carrier systems for microRNA delivery as well as potential therapeutic effects for cancer cells.
Melanoma is one of the most threatening skin cancers which when undetected leads to fatal results. There were 76,690 new cases and 9,480 deaths in U.S. alone. Early detection of melanoma is very crucial for treatment, as well as for the prevention of the metastatic cancer. The objective of the present study was to design a novel, easy-to-use, melanoma detection method. Here, Melan-A was chosen as the biomarker for melanoma cells. Because of its unique optical-electronics properties and bio-compatibility, gold nanoparticles are used for diagnosis and delivery of drugs. In this study, we created gold nano-needles and coated them with a specific monoclonal antibody to Melan-A to detect the melanoma cells. The conjugated golden nano-needles will bind to the protein, Melan-A, on the surface of the melanoma cells, and with its optical and colorimetric properties, the conjugated golden nano needles will change color for an optical positive reading visible to the naked eye.
Nanotechnology holds great promise to achieve revolutionary advances in virtually all aspects of medicine including in vitro diagnostics, bioimaging, targeted and externally-triggered therapy, image-guided surgery, and regenerative medicine. Multifunctional nanostructures that enable targeted and externally-triggered delivery of therapeutic agents along with non-invasive tracking and monitoring of the therapy process are a holy grail in nanomedicne. Here we report a novel multifunctional plasmonic nanorattle that enables externally-triggered and highly localized combination therapy and non-invasive tracking of therapy process using surface enhanced Raman scattering (SERS). Nanorattles are comprised of ultrabright SERS probes with two Raman reporters, precisely engineered to track the therapy process non-invasively. We demonstrate that a combination of photothermal ablation of the plasmonic nanorattles and triggered release of chemotherapeutic drug from the nanorattles can induce locoregional death of cancer cells in vitro. The completion of therapy process is indicated as a “Raman signal flip” between the two reporters of the SERS probe. Simple yet powerful approach to non-invasively monitor the therapy process serves as a new tool for precise control over the therapy process using plasmonic nanostructures.
Nanotechnology holds a great promise to overcome the current challenges in treatment of diseases such as cancer and neurological disorders by providing highly specific and efficient delivery vehicles. To date, many polymeric and lipid-based delivery systems have been developed and examined for delivery of drugs, proteins, and genes. Recently, hybrid lipid-coated polymer nanoparticles (LCPNs) that combine the advantages of biomimetic lipid membranes and solid polymeric nanoparticles have attracted a lot of attention since these vehicles offer excellent loading capacities, tunable and sustained release profiles, highly specific delivery, and outstanding serum stabilities. These LCPNs have, therefore, been explored for delivery of a number of therapeutics and vaccines to different sites of the body. In this study, we aim to develop LCPNs that enable efficient delivery of therapeutics to the brain to ultimately target brain tumors. Many of therapeutic compounds currently used for treatment of brain cancer or neurological disorders are hydrophobic and hence, require appropriate delivery systems to effectively reach their target site. Here, we investigate the potential of LCNPs for encapsulation and release of a hydrophobic cargo, curcumin that has been applied for treatment of diseases such as cancer and Alzheimer&’s disease (AD). Curcumin is a member of the ginger family and a traditional medicine in countries such as India and China. This natural compound is known to have anti-oxidant, anti-inflammation, anti-amyloid, and anti-tau hyperphosphorylation properties that make it an appealing candidate to tackle cancer and amyloidogenic diseases such as AD. The application of this compound has, however, remained limited due to its poor solubility in aqueous solutions, which in turn limits its bioavailability. In this work, we applied FDA-approved biodegradable polymer, poly (lactide-co-glycolide) acid (PLGA), to encapsulate curcumin and coated these particles with lipid membranes that would carry specific targeting ligands for efficient delivery to the brain tumors. We used nanoprecipitation technique to fabricate curcumin-loaded PLGA nanoparticles and investigated the effect of different parameters such as polymer and drug concentration on particle properties. Resultant drug-loaded nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). We then encapsulated these nanoparticles within a lipid membrane coating, and evaluated their properties such as size and zeta potential compared to bare PLGA nanoparticles. Finally, we examined and compared the encapsulation efficacy and release profile of curcumin in PLGA and lipid-enveloped PLGA nanoparticles. These LCNPs will next be decorated with tumor-specific ligands for targeted delivery to the brain tumors. Long-term goal of this work is to employ these LCPNs for delivery of anti-cancer agents to brain tumors in vivo.
Over the last two decades, the use of bioderived nanomaterials have gained much traction where in this field, viruses represent a unique, self-assembling multifunctional platform. Viruses have, for instance been used as biological templates, delivery vessels, and nanoscale catalysts for chemical reactions or materials synthesis. In this work we use of a virus-like particle as a multivalent platform to demonstrate vitro targeted photodynamic therapy (PDT) experiments toward breast cancer. PDT is an effective treatment for many cancer types, yet the current class of photosensitizers suffers from lack of tissue specificity, often leading to unwanted side effects. By loading photosensitizers into a targeted nanodelivery vessel, potential side effects resulting from systemic photosensitizer accumulation could be avoided. Additionally, delivering a relatively high concentration of photosensitizers in a confined nanoscale volume can increase the amount of localized singlet oxygen concentration, leading to increased PDT efficacy. In this work, nanoscale drug delivery vessels are formed via nucleotide-driven packaging of cationic porphyrins inside MS2 bacteriophage capsids, followed by attachment of G-quadruplex DNA aptamers to the capsid exterior via chemical conjugation. Prior work has demonstrated that approximately 250 porphyrins can be loaded into each capsid, and capsid-loaded porphyrins retain their capability to generate singlet oxygen upon photoexcitation with 632 nm light. The G-quadruplex DNA aptamer targets nucleolin, which is often overexpressed on the surface of many cancer cells. We show that approximately 80 DNA aptamers were conjugated to the surface of each capsid, previously loaded with the cationic porphyrin TMAP. Cell viability was assayed with the MTT assay and Live/Dead staining where targeted PDT experiments showed that MCF-7 human mammary adenocarcinoma cells were selectively killed, whereas normal human mammary epithelial MCF-10A cells remain unaffected.
Our recent studies have proposed that layer-by-layer (LbL) assembly of polyelectrolytes on nanoparticles provides a promising drug delivery system. These studies have investigated the impact of different film architectures on the nanoparticle surface, elucidating key control variables necessary to generate a serum-stable particle as well as the effect of terminal layers on the pharmacokinetics of the nanocarriers. The current work is aimed to incorporate siRNA into nanoparticles through layer-by-layer assembly. In combination with liposomal doxorubicin, this work presents a combinatorial delivery design for more efficacious treatment of cancer.We first screened a library of both natural and synthetic polycations for optimal loading of siRNA/polycation LbL films on the nanoparticles. The physicochemical properties of these LbL nanoparticles were carefully examined, which included the size, surface charge, polydispersity, siRNA loading, film stability and N-to-P ratio. We demonstrated that incorporation of polycation-siRNA layers on nanoparticles were colloidally stable and resulted in uniform coatings, as indicated by an approximately 5 nm increase per layer in hydrodynamic size. In GFP-expressing breast cancer MDA-MB-468 cells, a dose-dependent gene silencing was seen for the candidate LbL nanoparticle. The candidates yielding minimal cytotoxicity and ideal release behavior were selected to demonstrate the systemic delivery of siRNA for TNBC targeting. In these studies, a single dose of the siRNA-loaded LbL nanoparticles that target a luciferase protein were intravenously injected into the nude mice with luciferase-expressing MDA-MB-468 cell xenografts. Five days post-injection, the treatments significantly decreased the target gene expression in the animal tumors, compared to the control of a sequence-scrambled siRNA treatment. The half life of the LbL nanoparticles was found to be 27 hours in the blood and a significant tumor accumulation effect was seen. These nanoparticles did not elicit any escalated level of inflammatory cytokines in the serum. To further assess the combination delivery, we incorporated the siRNA LbL films onto liposomes loaded with chemotherapy drugs, such as doxorubicin or cisplatin. A staggered release profile was seen for the two components in physiological buffer. A co-delivery of doxorubicin with siRNA that targets a resistance reverse gene was tested both in vitro and in vivo (subcutaneous and orthotopic xenograft) human triple negative breast cancer models. The combinatory delivery demonstrated an improved efficacy of the chemotherapy drug, compared to the single component treatment.This work presented a novel delivery platform for combinatory therapeutics using LbL nanoparticles. This platform is easy-to-fabricate, modular and controlled for improved biostability and delivery of RNAi and chemotherapeutics. The study here also presents a potential new treatment for treat triple negative breast cancer.
Poor experimental models that recapitulate the bone metastatic environment have led to poor options for breast cancer patients with bone metastatic lesions that are usually incurable. There is also a clinical problem of patient-to-patient variability in rates of disease progression and metastasis due to differences in the cancer cells, but also in the osteoclasts recruited in the metastatic niche to assist local proteolytic remodeling necessary for osteolytic lesion establishment, primarily through secretion of cathepsin K, the most powerful human collagenase. The relative contributions of each and synergism between the two in altering the biochemical and biomechanical properties of the colonized bone are difficult to parse with animal models. To quantify the relative contributions of breast cancer cells and osteoclasts in bone resorption, we have been developing engineered bone microenvironment tissue surrogates by adapting a poly(ester urethane) urea system embedded with microbone particles. When seeded with breast cancer cells and/or osteoclasts isolated from human subjects or cell lines, this can provide temporal, multiscale reporters of bone resorption that can be measured non-destructively: 1) collagen degradation measured by C-terminal collagen fragment release, 2) mineral dissolution by measuring calcium released with the calcium arsenazo assay, and 3) fluorogenic reporters of cathepsin activity with BODIPY-labeled extracellular matrix substrates. Additionally, osteoclast production of cathepsin K varies from patient to patient, which may be correlated with progression to metastatic disease. This study will report differences in bone resorption by osteoclasts isolated from different patients co-cultured with and without breast cancer cells quantified using these novel multiscale biomaterials as a model system to investigate synergism between metastasized breast cancer cells and recruited osteoclasts to osteolytic lesion formation.
Developing high-performance, multifunctional nanodevices for cancer (“theranostics”) is a major direction in nanomedicine. Magnetic-plasmonic nanoparticles (NPs) have high potential for medical applications. High surface enhanced Raman scattering (SERS) signals due to suitably shaped plasmonic Au NPs can be used for biological sensing and medical imaging. On the other hand, magnetic Fe3O4 NPs can generate heat under an alternating magnetic field, enabling hyperthermia therapy. Furthermore, a thermo-sensitive material such as poly(N-isopropylacrylamide) (pNIPAm) can be used for drug delivery. Combining Au, a Raman reporter, Fe3O4, pNIPAm, drug and an antibody could form novel nanodevices with both diagnostic and therapeutic functions. HER2-positive breast cancer is a cancer that tests positive for a protein called human epidermal growth factor receptor 2 (HER2), which promotes cancer cell growth. In this investigation, drug-loaded, thermo-sensitive and magnetic-plasmonic Fe3O4@Au@pNIPAm hybrid NPs with HER2 targeting ability were developed (NP diameter: ~50 nm). HER-2/c-erbB-2/neu rabbit polyclonal antibody was conjugated to hybrid NPs for cancer cell targeting. Sensing of HER2-positive cancer cells was provided by high SERS signals that were enabled by hybrid NPs which acted as a SERS-active tag. These hybrid NPs could only be internalized by human breast cancer cells with HER2 (e.g., SK-BR-3 cells), but not by human breast cancer cells without HER2 (e.g., MCF-7 cells). Under a NIR laser or an alternating magnetic field, hyperthermia would occur, causing cancer cell death. Firstly, core-shell structured Fe3O4@Au NPs were made. A pNIPAm coating, which was incorporated with 4-MBA (a Raman reporter) and 5-Fluoroaracil (5-FU, an anti-cancer drug), was produced on Fe3O4@Au. pNIPAm had a lower critical solution temperature (LCST) of 32.3°C. In in vitro release tests, below LCST, only a small amount of 5-FU was released from hybrid NPs. At 42°C, 5-FU was released quickly from hybrid NPs. The viability of SK-BR-3 cells and MCF-7 cells after 48 hrs incubation with hybrid NPs (at various concentrations: 0 to 100 µg/mL) was examined using MTT assay. The hybrid NPs exhibited good biocompatibility even at the NP concentration of 100 µg/mL. Antibody-conjugated hybrid NPs as a stable SERS-active tag for HER2-positive cancer cells were demonstrated. SK-BR-3 cells were detected via strong SERS signals, whereas MCF-7 cells showed little SERS signals. NPs in the right size range accumulated preferentially at SK-BR-3 cells. TEM and LSCM analyses showed that hybrid NPs were successfully internalized by SK-BR-3 cells. In contrast, no cellular uptake of hybrid NPs was observed in MCF-7 cells which are HER2-negtive cells. The hybrid NPs also offered hyperthermia therapy for cancer.
Targeted delivery of a cytotoxic drug is beneficial to maximize the efficacy of the drug and reduce side effects associated with its delivery. Nanoparticulate-based drug delivery systems are being developed to control the release of drugs in the body, to protect the drugs from enzymatic or chemical degradation, and to attain organ- or tissue-targeted delivery. Studies have shown that biological sources of calcium carbonate (CaCO3) nanoparticles are highly porous, biocompatible, biodegradable, and have pH-sensitive properties. Such desirable properties make CaCO3 nanoparticles one of the best candidates for biological drug delivery systems. In this research, the CaCO3 nanoparticles were derived from egg shells using sonochemical and mechanochemical methods. 5-Fluorouracil (5-FU) is a well-known anti-cancer drug, which is commonly used for several cancer therapies. 5-FU is administered subcutaneously or intravenously to patients, which results in low patient compliance. CaCO3 nanoparticles encapsulated with 5-FU were combined with other excipients to create a tablet for colonic drug delivery. To protect the tablet from premature degradation within the gastrointestinal (GI) tract, it was coated with Eudragit S100. We hypothesize that the combination of Eudragit S100 and 5-FU loaded CaCO3 nanoparticles will give the tablet a sustained release, after it reaches a pH above 7.2 in the GI tract. Dissolution studies for the tablet were tested in conditions mimicking the body, first placed into 0.1 M of HCl for two hours and then into phosphate buffered saline (PBS) for five hours at 37°C at 141 rpm. The transit time was monitored in vivo within Sprague Dawley rats and found to be radiologically undetectable after 4 hours. Results show a sustained release for 5 hours in vitro, but due to the sphincter within the rat&’s stomach, the tablet was mechanically opened while trying to exit the stomach.
Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient but also nonspecific, thereby posing a major shortcoming in effective treatment of brain cancer. Photodynamic therapy (PDT) is a localized treatment modality, relying on both a photosensitizer and drug activation using a specific wavelength. The widespread use of PDT in brain tumor therapy has been partially hampered by non-targeted phototoxicity towards healthy tissue. The development of nanoparticles selectively targeted to cell surface receptors that can act as drug delivery vehicles is critical for improving the treatment and therapeutic responsiveness in inaccessible tumors, such as glioblastomas. Gold nanoparticles (Au NPs) provide an excellent platform with a surface that can be tailored to attach biomolecules for targeted drug delivery and biocompatible coatings that can efficiently encapsulate the hydrophobic photosensitizer drug, Pc 4, thereby reducing off-site cytotoxicity. In this study, we demonstrate a novel double targeted, noncovalent Au NP drug delivery agent, which selectively delivers drugs to brain tumors for PDT. These double-targeted Au NPs loaded with Pc 4 have been compared with previously studied single targeted Au NPs. Hydrophobic Au NPs have been cap exchanged with mono- and bi-functional PEG linkers. Specific targeting of the PEGylated Au NPs to glioma cells is achieved by coupling receptor-binding peptides to the carboxyl moiety of the bi-functional PEG linker. Subsequently, hydrophobic Pc 4 is adsorbed in the PEG corona (mono-functional linker) to form Pc 4 loaded and targeted Au NPs. Packaging of Pc 4 within the PEG core impedes leaching of the drug into the extracellular environment and improves circulation in vivo. UV-Vis absorption measurements indicate encapsulation of Pc 4 within the PEGylated Au NPs. Hydrodynamic diameter of these agents lies well within the limits needed to cross the BBB as determined by dynamic light scattering. In vitro cell uptake studies in glioma cell lines, LN229 and U87, which express differential patterns of the epidermal growth factor (EGF) and transferrin (Tf) receptor targets, show a significant increase in cellular uptake and intracellular localization for double targeted conjugates as compared to either single targeted Au NPs. Titration studies have been carried out in cells to optimize delivery; the optimal concentration for double targeted Au NPs is 500 nM, half of the current clinical standard observed for single targeted Au NPs over the same period of incubation. In vivo imaging utilizing real time, longitudinal fluorescence in mice shows notable accumulation of these agents in the tumor. Co-localization of the targeted Au NPs in regions overexpressing EGF and Tf receptors has been validated by immunohistochemistry. Future experiments involve activation of Pc 4 by PDT after delivery by the double-targeted Au NPs and monitoring tumor cell death.
Primary human immune T cells isolated and frozen at an early healthier stage can be restored, genetically modified for specificity, selectively expanded, and administered to the patient at a later morbid stage to exert the anti-tumor response. The response to this therapy during the clinical trials, Adoptive T Cell Therapy, remains inconsistent at best. In order to improve the reliability, efficacy and safety of the clinical trials it is important to image the biodistribution of these genetically modified T cells. This can determine if the T cells are homing to their tumor targets and can be used to modulate the T-cell-based drug load. Current methods to assess biodistribution of infused T cells include serial sampling from various tissues followed by quantitative PCR (Q-PCR) or flow cytometry; which is invasive, painful, and does not provide whole-body distribution. Magnetic Resonance Imaging (MRI) has been used to image cells in vivo, it requires the approximate location of cells to be already known. We hypothesized that if T cells can be labeled with PET-MRI imaging agents, this initial positioning of cells can be provided by Positron Emission Tomography (PET) at high-sensitivity. MRI can then be used to scan these areas and report on anatomically correlated biodistribution of adoptively transferred T cells at high-resolution.Previously frozen peripheral blood mononuclear cells were thawed and genetically modified with Sleeping Beauty transposon/transposase system to express CD19-specific chimeric antigen receptor (CAR) and firefly luciferase (ffLuc) enzyme. CD19-specific-CAR+ffLuc+ T cells were selectively expanded on artificial antigen presenting cells and labeled with super paramagnetic iron oxide nanoparticles conjugated to fluorescent probe and positron emitter (SPION-FL-64Cu). SPION retention in T cells was studied using flow cytometry and the internalization into the cytoplasm was verified using confocal microscopy. Iron content in a single T cell was determined by inductively coupled plasma mass spectrometer. Effect of SPION and 64Cu on cell viability was assessed by the ffLuc enzymatic activity. MRI signal was obtained from the series of diluted and homogenously suspended T cell phantoms. Chromium release assay and live-cell time-lapse imaging was used to assess the in vitro tumor targeting capability of CD19-specific-CAR+ffLuc+SPION+ T cells.Our approach builds upon ongoing clinical trials for CD19+ B-cell malignancies and uses an approach that can be readily undertaken in compliance with current good manufacturing practice for Phase I/II trials.
Glioblastomas shed large quantities of small, membrane-bound microvesicles (MVs) into the circulation. While these hold promise as potential biomarkers of therapeutic response, there remain hurdles to their identification and quantitation. By tagging MVs with target-specific magnetic nanoparticles in a dedicated microfluidic platform and detecting them through a miniaturized nuclear magnetic resonance system, we develop a highly sensitive and rapid analytical technique for profiling circulating MVs directly from blood samples of glioblastoma patients. Compared with current standard assays (e.g., Western blotting, enzyme-linked immunosorbent assay and flow cytometry), this integrated system has a much higher detection sensitivity, and can differentiate glioblastoma multiforme (GBM) MVs from non-tumor host cell-derived MVs. The system further showed that circulating GBM MVs could serve as a surrogate for primary tumor by reflecting its molecular signature and a predictor of treatment-induced changes. We expect that this converging nanotechnology platform would have a wide range of applications, providing both an earlier indicator of drug efficacy and a potential molecular stratifier for human clinical trials.
Magnetic drug eluting microspheres will offer the direct visualization and deliver the anticancer drugs to reach the target site and maintain sufficient therapeutic activity in transcatheter-targeted therapy of hepatocellular carcinoma (HCC). 34 um drug eluting magnetic alginate microspheres containing magnetic clusters were successfully prepared by employing a microfluidic system. Sustained drug release rate was achieved with the incorporation of magnetic clusters that can be used for maintaining therapeutic concentrations in the tumor regions. Strong MR signals of the magnetic microspheres in phantoms were measured in T2 and T2*-weighted images. The MR visible drug eluting magnetic microspheres were successfully injected through the hepatic arterial targeted approach in HCC rat models, and the targeted magnetic microspheres were monitored on the HCC tumor region in vivo MR imaging and confirmed with histological analysis. These microspheres offer the potential benefits of visibility with MR imaging and controlled release of chemotherapeutic agents into the HCC tumor bed in an interventional transcatheter targeted therapy.
Nanomedicine increased rapidly with nanotechnology over the last decade, especially with the development and application of nanomaterials in different areas. Despite the rapid progress and acceptance of nanomedicine, the potential effects of nanomaterials in biological systems due to prolonged exposure at various levels of concentration, has not been established. Despite these obstacles, there is great interest in understanding their interactions in more specific details for nanotoxicity and also for the development of new theranostic agents. Due to the complexity of the mechanisms involved in the interactions between nanomaterials and biological systems, biophysical aspects are difficult to be investigated, especially in natural samples and in real time. In this study we introduce a methodology to evaluate the interactions between nanoparticles and phospholipid membrane models, using the Langmuir technique. Our main goal was to elucidate the biophysics interaction of nanomaterials in cell membranes, at the molecular level. The penetration of nanoparticles into lipids monolayers was studied using Langmuir techniques as kinetics absorption and surface pressure measurements, spectroscopy frequency generation (SFG) and also by flow cytometry with cancer and health cell lines. In the first system, we evaluated the interaction between single-walled carbon nanotubes (SWCNTs) and dipalmitoylphosphatidylcholine (DPPC) lipids monolayers. The penetration of SWCNTs/polyamidoamine dendrimer nanocomplexes into DPPC monolayers was pronounced, as revealed by adsorption kinetics and surface pressure measurements, suggesting that SWCNTs were able to interact even at high surface pressure values, ~30 mN/m. In the second system, we observed the interaction between silver nanoparticles (AgNp) and cardiolipin monolayers. In both cases, the results confirm that the presence of the nanomaterial affects the packing of the synthetic membranes. The incorporation of the nanomaterials into the monolayers was pronounced, as revealed by the shift in the molecular area to higher values, compared to the values obtained for pure lipids. Such investigations may be of great importance to understanding the toxicity of nanomaterials at the molecular scale and bringing important benefits to the nanomedicine development.
The study focuses on fibroblast-derived materials obtained from human renal cell carcinoma (RCC) as an example to attain in vitro mimicry of the effects that stromal extracellular matrix (ECM) imparts on tumorigenesis. Surgery and targeted therapies have limited impact on survival in patients with advanced RCC, and metastasis to distant organs account for the majority of deaths. One of the striking features of RCC is that it is characterized by a fibrous stromal reaction producing an altered ECM that directly intercalates with the cancerous epithelia and plays a functional role in the disease progression. Much of our work is based on the fact that stromal-tumor cell interaction dynamics are most readily studied in in vivo-mimetic three-dimensional (3D) models. Among the most promising of these are in vitro 3D stromal systems developed by our group in which tumor cells are plated within tumor-associated ECM derived from fibroblasts harvested from surgical tissue samples comprised of patient-matched normal and tumor-associated fibroblasts. We have demonstrated the architectural and physiological relevance of this system and found that mesenchymal stromal cells and matrices are gradually modified during epithelial tumor progression in vitro and in vivo. Importantly, we have also demonstrated that stromal activation levels are clinically predictive in RCC.Here we show that ECMs produced by normal fibroblasts are restrictive, while matched tumor-associated ECMs induce increased RCC tumorigenic responses including growth and resistance to apoptosis as well as invasion. An unbiased gene expression array was conducted using RNA obtained from RCC cells cultured within normal or tumor-associated ECMs. Classic RCC signaling pathways were evident and, in addition, proteins previously unsuspected to play important roles in stromal regulation of RCC progression were identified. Using our mimetic 3D stromal system, we demonstrate that these novel proteins play important roles in stromal induced RCC tumorigenesis. Moreover we demonstrate the clinical importance of our discovery using a human RCC patient cohort. We strongly believe that, by using the mimetic stromal 3D system, we can facilitate uncovering mechanisms responsible for tumor-associated ECM induced tumorigenesis and metastasis. These approaches could result in the identification of novel stromal regulated tumoral targets that when blocked could serve as novel therapeutics. Funding was provided by the Commonwealth of Pennsylvania, the Keystone Program in Personalized Kidney Cancer Therapy, a Temple-FCCC Nodal Grant Award and NCI/NIH grants CA113451 and CA06927.
Here we will describe the first functional use of recently introduced ultrabright fluorescent mesoporous silica nanoparticles, which are functionalized with folic acid, to distinguish cancerous and precancerous cervical epithelial cells from normal cells. The high brightness of the particles is advantageous for fast and reliable identification of both precancerous and cancerous cells. Normal and cancer cells were isolated from three healthy women and three cancer patients. Three precancerous cell lines were derived by immortalization of primary cultures of normal cells with human papillomavirus type-16 (HPV-16) DNA. We observed substantially different particle internalization by normal and cancerous/precancerous cells after a short incubation time of 15 minutes. Compared to HPV-DNA and cell pathology tests, which are currently used for prescreening of cervical cancer, we demonstrated that the specificity of our method was similar (94-95%), whereas its sensitivity was significantly better (95-97%) than the sensitivity of those currently used tests (16%-82%).
Tracking of immune cells is important due to their crucial role in different pathological conditions including cardiovascular disease and cancer. Effective tracking of cells facilitates better understanding of disease progression mechanism and subsequent intervention. Moreover, using immune cells as transport vehicles for imaging and therapeutic agents can be beneficial in imaging and treating cancer in avascular regions. As biological tissues adsorb light in the visible spectral range, development of contrast agents with high optical cross section in the near infrared region is essential for cell tracking. Gold nanorods are attractive candidates for in-vivo tracking of multiple cell populations for several reasons - high optical cross-sections, tunable absorbance in the near-IR region, chemical inertness and biocompatibility. Furthermore, gold nanorods provide high imaging contrast in non-invasive photoacoustic imaging modality due to their strong optical absorption. However, optical spectra of gold nanorods broaden upon cellular uptake due to plasmon resonance coupling; this renders simultaneous imaging of multiple cell populations labeled with different nanorods difficult due to significant increase in spectral overlap. To address this problem, we have synthesized silica-coated gold nanorods with fluorescent dye embedded in silica matrix by either physical encapsulation or covalent linking approach and demonstrated that plasmon resonance coupling can be circumvented by the silica coating. Silica-coated nanorods with fluorescent dyes provide possibility for fluorescent imaging using animal models with implanted optical windows and also facilitate ex-vivo tissue evaluation. We demonstrated a good cellular uptake of silica coated gold nanorods by two macrophage cell lines with no cytotoxicity up to two days. Photoacoustic imaging of tissue-mimicking phantoms with inclusions containing different concentrations of nanorod-loaded macrophages showed linear increase in the photoacoustic signal with number of cells and a sensitivity of five cells per imaging kernel of a size given by 162 µm x 108 µm x 216 µm.
Biomimetic nanoparticles with enzymatic activities offer new possibilities for replacing a particular enzyme in patho-physiological settings. We have shown previously that we can mimic the enzymatic activity of vanadium haloperoxidases using V2O5 nanoparticles (Nature Nanotechnol 2012, 7, 530). Here we demonstrate the sulfite oxidase biomimetic activity of MoO3 nanoparticles which are extremely active for catalyzing the oxidation of sulfite to sulfate. A defect in the enzyme sulfite oxidase (SuOx) in humans leads to a devastating disorder resulting in early death. The typical disease expression involves neonatal seizures, dislocated ocular lenses, progressive encephalopathy and developmental delay with psychomotor retardation. To date no medical treatments for SuOx deficiency are known.Sulfite oxidase (SuOx) is one of three human enzymes that require molybdenum as inorganic cofactor. It is localized in the mitochondrial intermembrane space and the molybdenum center promotes the catalytic oxidation of sulfite to sulfate, the terminal reaction of the degradation pathway sulfur containing amino acids. Its biological function is detoxification of sulfur dioxide and sulfite to sulfate. SuOx is localized in the human body mainly in the liver and kidneys. MoO3 nanoparticles were surface functionalized with a bifunctional ligand that carries on one side am anchor group for covalent binding to the nanoparticle surface and on the other end a triphenylphosphin unit that aids crossing cellular lipid bilayers and that selectively targets the mitochondrial membrane where SUOX is located. Ligand functionalized MoO3 nanoparticles were assayed for their capacity to the mimic sulfite oxidase (SuOX) reaction with a standard SuOX assay that includes a defined amount of enzyme/nanoparticle, sodium sulfite and cytochrome c. In order to calculate the kinetic parameters (Michaelis-Menten kinetics) the concentration dependences (reaction rate vs. concentration) of the reaction partners (nanoparticle/sulfite/cytochrome c) were determined. The MoO3 nanoparticles were localized during cell uptake studies by confocal laser scanning microscopy (CLSM) with fluorescent markers attached to the particle and to the ligand. The nanoparticles accumulate inside the cell; mitochondria-localization of the functionalized MoO3 nanoparticles was performed with Mito Tracker Green, a dye that selectively stains mitochondria inside the cell, and a dye conjugated to the phosphine ligand. The intracellular SuOX activity of the functionalized MoO3 particles was demonstrated with a SuOX knockdown cell line.
Development of multidrug resistance (MDR) in cancer cells are major obstacles for effective cancer chemotherapy. Therapeutic strategies to overcome drug resistance should have a significant impact on the treatment of cancer. A new approach to overcome MDR is to use a co-delivery system that combines an anticancer drug and suppressors of cellular resistance in drug resistance cells. In this paper, a novel nanocarrier fabricated directly from an anticancer drug-camptothecin (CPT) is reported. Taking advantage of the strong hydrophobicity of the anticancer drug CPT, CPT molecules were conjugated to a hydrophilic oligopeptide, forming amphiphilic liposome-like structure. The prodrugs are able to condense nucleic acids and delivery the therapeutic nucleic acid drugs for MDR cancer treatment. In contrast to those delivery systems which anticancer drugs are embedded in or conjugated with inert nanocarriers, this drug-conjugates strategy using drugs as part of a carrier reduces the use of inert materials in carriers and increases drug loading content. As a proof of concept, the synthesis and characterization of one CPT-prodrug are demonstrated. Intracellular localization, cytotoxicity, gene expression and apoptosis of this drug conjugate against MDA-MB-231 breast cancer cells are examined.
Directly applying therapeutic drugs to patients suffers from the intrinsic limitations of these small molecules including poor physiological stability, non-specific targeting, and low cell membrane permeability. So the design of drug carriers to deliver drugs to specific targets is one of the potential strategies to overcome these limitations. In this work we developed a pH-responsive drug delivery system (DDS) with high cellular uptake efficiency by utilizing a nano-sized metal organic framework (MOF), which possesses the advantages of both organic- and inorganic-based DDSs. The studies show that the MOF nanospheres are uniform 70 nm crystals with single-crystalline structure. The small molecules including fluorescein molecules and anticancer drug camptothecin molecules were encapsulated inside of MOF frameworks by simply introducing small molecules in the MOF synthesis solution. Then we incubated the molecule-encapsulated MOF nanospheres with MCF-7 breast cancer cell line. The confocal laser scanning microscopy study and the MTT assay data show that these molecule-encapsulated MOF nanospheres are ideal for drug delivery. The nanospheres are stable in physiological conditions and the cytotoxicity of the MOF carrier is low. The cellular uptake efficiency is high due to the 70 nm size. The drug molecules can be released after the cellular uptake because of the pH-responsive dissociation of MOF frameworks. To demonstrate the versatile potential of this MOF system, iron oxide nanoparticles were also encapsulated to the molecule-loaded MOF nanospheres, thereby endowing magnetic features to the nanospheres.
We prepared organically modified silica (ORMOSIL) nanoparticles with internal functional groups and mesoporosity, suitable for the incorporation of modalities for both MRI imaging and cancer treatment by neutron capture therapy using boron-10 and gadolinium-157 nuclei. These modalities were incorporated by preparing ORMOSIL nanoparticles with reactive functional groups throughout the nanoparticle body, followed by their conversion into the metal chelating and boron-containing moieties inside the nanoparticles. Furthermore, we incorporated pH-sensitive carbamate linkages into the particles to render them biodegradable, and controlled the nanoparticle porosity by templating the particles with tannic acid. This talk will describe in detail the preparation, characterization and properties of the resulting theranostic agents.
Breast cancer is the second leading cause of cancer mortality in the US. However, only metastases are responsible for these deaths. Metastasis occurs when cancerous cells adopt a migratory, invasive behavior and move to the circulatory system to travel elsewhere in the body. If cell adhesion to their original niche were improved, the metastatic potential of these cells would likely decrease. We have previously found that high aspect ratio carbon nanoparticles can decrease cell mobility; therefore, we hypothesized that they might also affect adhesion. We investigated the ability of high aspect ratio nanoparticles to change adhesive behavior on collagen substrates using carboxylated multi-wall carbon nanotubes (MWNT-COOH) and the control groups of carbon black and collagen alone. Each nanoparticle type was evaluated at 5 mu;g/mL, 10 mu;g/mL, 20 mu;g/mL, 25 mu;g/mL, 30 mu;g/mL, 50 mu;g/mL, and 100 mu;g/mL. The nanoparticles were incorporated into the collagen and then allowed to dry to form a coating. Because adhesion is mediated through cadherin proteins and the focal adhesion complex, we evaluated E-cadherin, N-cadherin, and α-catenin, which is part of the focal adhesion complex. We measured the cellular response to the different coatings using RT-PCR, adhesion and proliferation assays, and immunofluorescent staining. We evaluated cells with a range of metastatic potential to see if any observed effect tracked with cell characteristics. MCF10A, MCF7, and MDA-MB-231 cells were tested. These represent normal breast cells, endothelial breast cancer cells, and triple negative breast cancer cells respectively. We found that adhesion significantly increased at the 20 mu;g/mL, 25 mu;g/mL, and 30 mu;g/mL concentrations with the carboxylated MWNT, but no change was observed at any concentration of the spherical, low aspect ratio carbon black nanoparticle. Within the MWNT data, the increase in adhesion was much larger for the breast cancer cells than the normal breast cells. Both nanoparticles types were generally non-cytotoxic, though both types reduced cell viability to roughly 60% of the control only at the highest concentration tested (100 mu;g/mL) in the triple negative cell line MDA-MB-231. E-cadherin, a measure of normal adhesion, also increased. We conclude that MWNT may be able to be applied to increase cancer cell adhesion, but the effect is dependent on both concentration and the aspect ratio of the carbon nanoparticle.
Over the several decades, the major huddles of the nanoparticles as drug carriers and diagnostic agent for tumor theragnosis were unintended burst release of loaded drugs and their poor physical stability during blood circulation. To overcome these issues, we developed photo-crosslinked hyaluronic acid nanoparticles (c-HANPs) with high stability and tumor targetability. c-HANPs were prepared via UV-triggered chemical crosslinking of the HA derivative bearing the acrylate group. The size and morphology of c-HANPs were not significantly changed by chemical crosslinking. However, c-HANPs showed high stability in the physiological buffer condition and released the loaded anti-cancer drug, paclitaxel (PTX), in a sustained manner. It is noteworthy that the release rate of PTX from c-HANP was significantly increased in the presence of hyaluronidase, an enzyme abundant at the tumor tissue and intracellular compartment of tumor cells. The in vitro results indicated that c-HANPs were rapidly taken up by the tumor cells via the CD44 receptor-mediated endocytosis. In the non-invasive optical imaging test, c-HANPs showed higher tumor-accumulation than uncrosslinked HANPs, suggesting that improved stability of c-HANPs enabled their long circulation in the blood stream. Owing to the sustained release of PTX and higher tumor-accumulation, therapeutic efficacy of PTX-loaded c-HANPs was higher than those of PTX-loaded uncrosslinked HANPs and free PTX.Overall, c-HANPs with high stability showed the promising potential as tumor-targeting nanocarriers.
Hyaluronic acid (HA), a naturally occurring polysaccharide, has been widely used for biomedical applications because of its excellent biocompatibility and biodegradability. Methotrexate (MTX), an analogue of folic acid, has been extensively used for the treatment of rheumatoid arthritis (RA). However, the clinical applications of MTX have been limited by severe side effects, owing to its cytotoxicity to normal cells in the body. In an attempt to address this issue, we have synthesized pH-sensitive HA-MTX conjugates through the esterification reaction. The structure of HA-MTX conjugates was characterized using 1H-NMR and UV-vis spectrophotometer. MTX was released in a sustained manner in physiological solution (pH 7.4), whereas its release rate dramatically increased at the mildly acidic condition, mimicking the pathological environment of RA. When activated RAW264.7 cells as the model cell line involving the pathogenesis of RA were treated with Cy5.5-labeled HA-MTX conjugates, strong fluorescence signals were observed in the cytosol of cells, indicating efficient cellular uptake of HA-MTX conjugates by receptor-mediated endocytosis. The in vivo biodistribution and therapeutic efficacy of HA-MTX conjugates were investigated using a collagen-induced arthritis mouse model. These results imply that HA-MTX conjugates can be an efficient formulation for treatment of rheumatoid arthritis.
Novel organic and inorganic nanostructures for localized and externally-triggered delivery of therapeutic agents at a target site have received immense attention over the past decade owing to their enormous potential in treating complex diseases such as cancer. Gold nanocages, a novel class of hollow plasmonic nanostructures, have been recently demonstrated to serve as carriers for the delivery of payload with external trigger such as light or ultrasound. In this Letter, we demonstrate that surface enhanced Raman spectroscopy (SERS) can be employed to non-invasively monitor the release of payload from these hollow plasmonic nanostructures. The large enhancement of electromagnetic (EM) field at the interior surface of these nanostructures enables us to monitor the controlled release of Raman-active cargo from nanocages. Considering that SERS can be excited and collected in near infrared (NIR) therapeutic window, this technique can serve as a powerful tool to monitor the drug release in vivo, providing additional control over externally triggered drug administration.
The biomedical applications of gold nanoparticles are expanding, in particular in photothermal therapy and as radiosensitizers in internal radiotherapy. Injections of gold nanoparticles could be advantageously monitored in vivo by using computed tomography (CT) as well magnetic resonance imaging (MRI). [2, 3] For this, it is necessary to label gold with a paramagnetic element such as manganese. However, the synthesis of gold/manganese nanoparticles (Au-Mn NPs) is not straightforward, requiring several steps that include gold nanoparticles synthesis by conventional colloidal chemistry techniques, purification of the nanoparticles and functionalization with manganese ions.  On the other hand, Pulsed Laser Ablation in Liquids (PLAL) has been increasingly explored as an alternative technique for nanoparticles synthesis. This preparation method allows a single-step production of pure and electrostatically stabilized nanoparticles of diverse materials without the addition of stabilizing ligands. In the present study, we have synthesized Au-Mn NPs. These particles were obtained upon ablation of a gold/manganese powder target in water with a KrF excimer laser. The synthesis of Au-Mn particles was demonstrated by UV-vis spectroscopy, EDS, XRD and XPS. TEM observation of Au-Mn particles showed dispersed nanoparticles with a size distribution of 5 nm ± 1 nm. The newly synthesized Au-Mn nanoparticles are promising for characterisation and application as a MRI/CT contrast agent, however further physico-chemical analysis will be performed to elucidate the binding mechanism and distribution of manganese onto the surface of Au NPs and the stability of this linkage.  E. C. Dreaden, A. M. Alkilany, X. Huang, C. J. Murphy and M. A. El-Sayed, Chemical Society reviews, 2012, 41, 2740-2779. V. W. K. Ng, R. Berti, F. Lesage and A. Kakkar, Journal of Materials Chemistry B, 2013, 1, 9. C. Alric, J. Taleb, G. L. Duc, C. Mandon, C. Billotey, A. L. Meur-Herland, T. Brochard, F. Vocanson, M. Janier, P. Perriat, S. Roux and O. Tillement, Journal of the American CHemical Society, 2008, 130, 5908-5915. S. H. Murph, S. Jacobs, J. Liu, T. C. Hu, M. Siegfired, S. Serkiz and J. Hudson, J Nanopart Res, 2012, 14, 1-13. S. Barcikowski and G. Compagnini, Physical chemistry chemical physics : PCCP, 2013, 15, 3022-3026.
A unique Janus nanoassembly comprised of polystyrene/Fe3O4@silica was developed via a one-pot miniemulsion synthesis. Distinct dual surface functionalities were achieved simultaneously for effective conjugation of functionally different chemical moieties. Folic acid as the targeting ligand was immobilized on the carboxyl decorated polystyrene matrix for enhancing tumor-selective targeting and internalization. Doxorubicin was attached via a pH-sensitive hydrazone bond to the silica surface to enable a controlled, stimulus-induced drug release after folate-mediated endocytosis under mild acidic conditions in endosomal compartments. More importantly, Fe3O4 nanoparticles embedded in the silica half shell provide possibilities for multimodal imaging and hyperthermia-induced sensitization of tumor cells. Consequently, this novel Janus structure with dual surface functionalities can serve as an innovative drug delivery platform for multidimensional cancer therapy.
Breast cancer reoccurs in approximately 20 percent of all patients between 5 and 10 years after successful treatment of the primary tumor.1 Many of these recurrences are believed to arise from tumor cells at distant metastatic sites that have reactivated after long periods of dormancy. Remodeling of the extracellular matrix (ECM) at these sites over time is hypothesized to trigger the release of the tumor cells from dormancy, enabling the formation of new lesions distant from the original primary tumor site.2 Two-dimensional (2D) and three-dimensional (3D) in vitro culture models have been developed to recreate some of the interactions between cancer cells and their microenvironment. In 2D, fibronectin (FN) and fibroblast growth factor 2 (FGF2) has been shown to induce dormancy,3 whereas in 3D, FN has been shown to inhibit dormancy and activate tumor cells through integrin β1 signaling.4 Lack of mechanical property control and differences between biochemical composition of these model systems and the native ECM make it challenging to identify key ECM signals that promote dormancy or activation. Additional studies are needed to understand how dynamic changes in the ECM at specific metastatic sites regulate reactivation after dormancy.Here, we are designing a hydrogel-based 3D culture platform to mimic critical mechanical and biochemical properties of different metastatic site tissues including the bone marrow, liver, and lungs. Poly(ethylene glycol)(PEG)-based hydrogels have been polymerized by different radically-initiated thiol-ene chemistries using PEG-4-thiol, prepared by modification of a four-arm PEG-OH monomer with thiols,5 and peptides modified with enes of different reactivities to tune reaction times. Hydrogels formed between 5-10 minutes after the application of cytocompatible doses of UV light (10 mW/cm2, 365 nm)6. Mechanical properties of the hydrogels were measured by rheology and the elastic moduli of soft metastatic site tissues were achieved (~0.5-5 kPa)7. MCF-7 breast cancer cells encapsulated within these PEG hydrogels are viable with the incorporation of a FN-based cell adhesion peptide (RGDS) to allow cell attachment to the bioinert hydrogel matrix. The highly tunable nature of these hydrogels will permit the incorporation of other peptide sequences that mimic various ECM proteins present in metastatic sites, such as laminin and collagens I and IV. These results demonstrate that a PEG-based hydrogel can be used as a physical model of the metastatic ECM and to investigate the role of the ECM in regulating cancer dormancy and recurrence.1 A Brewster, et al. J Natl Cancer Inst 2008, 100, 1179-1183.2 J Townson, et al. Cell Cycle 2006, 5, 1744-1750.3 J Barrios, et al. Cancer Microenviron 2009, 2, 33-47.4 D Barkan, et al. Cancer Res 2008, 68, 6241-6250.5 B Fairbanks, et al. Macromolecules 2011, 44, 2444-2450.6 B Fairbanks, et al. Adv Mater 2009, 21, 5005-10.7 I Levental, et al. Soft Matter 2006, 91, 1-9.
Understanding the dynamics of magnetic nanoparticles is of great interest to research in biophysical sensing, magnetic recording, and thermal cancer therapies. The behaviors of these nanoscale dipolar magnets are well studied by Langevin and Fokker-Planck approaches. At the same time, many experiments have been accomplished to study the dynamics through magnetic spectroscopy. This technique examines the energy states of the particles in order to infer microenvironmental parameters including viscosity, temperature, and biochemical binding. To access the external environment, we require the particle to physically rotate, the so-called Brownian regime. However, a temperature for the nanoparticles can also be meaningful in the Néel regime, for which the particle magnetization changes direction internally. Recently, a new method of magnetic spectroscopy has been proposed that is especially pertinent to techniques that use strong, fixed frequency fields like those used in nanoparticle hyperthermia treatment of cancer. To determine the correct and safe dosage of heat it is essential to monitor the temperature of nanoparticles during hyperthermia. Here we examine the possible uses of this new technique to measure the local temperature of the particles using a pickup coil fully decoupled from the excitation field. We use stochastic dynamical Langevin equation simulations to motivate the problem, and demonstrate agreement from preliminary experimental data.
Photodynamic therapy (PDT) has been considered as one of promising strategy for various cancer treatments. The function of PDT depends on a special drug known as photosensitizer (PS) that can be activated by light at a particular wavelength to destroy the targeted cancer tissues, while leaving the nearby normal cells unharmed. Although the hydrophobicity of PS molecules is preferable for allowing them to localize towards cancer tissues, it often leads to low solubility of the PS drug molecules in blood stream, hampering their availability at cancer sites for PDT. To overcome this issue, the PS drugs are often encapsulated in a specialized carrier to improve the solubility of the PS, thus prolonging the drugs circulation while still maintaining their preferential accumulation at the tumor sites for PDT. In this study, a new type of core-shell polymeric carrier to deliver meso-tetra(p-hydroxyphenyl)porphin (p-THPP) anti-cancer PS drug for PDT has been successfully developed from biodegradable and biocompatible polyesters polyhydroxyalkanoates (PHAs). Here, three different types of PHA known as poly(hydroxyburytrate-co-hydroxyvalerate) or P(HB-co-HV) having similar molecular weights, but varied in their %HV mole compositions were synthesized and purified from Cupriavidus necator H16. These PHAs copolymers were used for making nanoparticles in combination with two different sets of polyvinylalcohol (PVA) shell polymers via a modified emulsification-diffusion method. The different formulations of p-THPP-loaded PHA nanoparticles were subjected to various characterizations. The sizes of all p-THPP-loaded PHA nanoparticles were ranging from 191 to 211 nm in diameter. The % drug loadings of PHA nanoparticles was also optimized to be ranging from 4% to 9% with % entrapment efficiencies ranging from 41% to 47%. The preliminary in vitro photocytotoxicity against HT-29 colon cancer cells showed that the p-THPP-loaded P(HB-co-65%HV) nanoparticles could lead to almost 70% cell death at the equivalent drug dose of 8 µg/ml, after 6 h pre-incubation and the irradiated light dose at 6 J/cm2, which was comparable to the effect of free p-THPP drug at higher concentration (i.e. 8 µg/ml). Thus far, this is the first report employing PHA nanoparticles as a PS drug carrier for PDT that also demonstrates their potentially beneficial effect of slow and controlled-release PS drugs for PDT.
The process of oncogenesis involves multiple mutations leading to immortalization, reduction in growth factor requirements, and enhanced potential for tumor formation and metastasis. Using in vitro models, we have previously shown that compared to normal keratinocytes, keratinocytes isolated from squamous cell carcinomas (SCC12b and SCC13) have two fold reductions in cell moduli and two fold increases in the moduli of their extracellular matrices measured by scanning modulation force microscopy (SMFM). The transformed keratinocytes also showed loss of lecithin retinol acyltransferase (LRAT), an enzyme that catalyzes the synthesis of retinylesters from retinol (vitamin A). To determine whether the changes in cell and matrix mechanics occur with the reduction in LRAT expression (qRT-PCR), we measured each parameter in nonstumorigenic, immortalized keratinocytes. We found that cell and matrix mechanics were comparable to SCC12b and SCC13, but LRAT expression was comparable to that of normal keratinocytes. Thus, changes in cell and matrix mechanics are earlier events in the transformation process. These properties are being used to develop adjunctive diagnostics to identify precancerous lesions and aid in cancer staging.
Cancer is a major human health problem worldwide and it is the second leading cause of death in USA . Breast cancer is the most fatal disease especially for women worldwide. Tamoxifen (TMX), a non-steroidal antiestrogen and a selective estrogen receptor modulator, has been the clinical choice for the antiestrogenic treatment of metastasis or advanced breast cancer for more than 20 years . This study aimed to investigate the effect of TMX and its encapsulation into a biodegradable nanoparticles prepared from copolymer of ethyl acrylate, methyl methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups (EUDRAGIT® RL 100), this copolymer have obtained considerable interest in recent years for their use as a delivery vehicle for various pharmaceutical agents. This new drug delivery system (TMX-NP) was designed to protect the TMX from environmental and to promote a sustained drug delivery profile of the drug. The size and entrapment efficiency were evaluated at different preparation protocols. TMX showed a maximum entrapment efficient of 23% in the proportion of 1,5 g of polymer and association to the fluorescence probe using a emulsion method. The size measurements were compared with the empty nanoparticles and showed values of TMX-NP = 166.4± 32 nm, and empty NP = 273± 50 nm. The zeta potential of particles was +65 and +38 mV for TMX-NP and empty NP respectively. The new drug delivery system posses some features parameter that allow it to be tested in biological samples such as cancer cells (4T1). Cytotoxicity assay shows that while the empty nanoparticles exhibit no significant cytotoxicity against 4T1 cells, 80% of these cells were killed after incubation for 24 hours with TMX-Nanoparticles (1mM).References:  Zhang et al. Biochemical and Biophysical Research Communications (2012), 417 679-85Sahana et al. International Journal of Nanomedicine (2010), 5 621-630Acknowledgements:We are grateful to CNPq, CAPES, FINATEC, Fundaccedil;atilde;o Lemann and FAPDF for financial support of this research.
Recently, nanomaterials-based imaging and sensing have gained a lot of interest in the field of various biological and medical systems. For sensitive and selective recognition of target biomolecules, it is very important to control the orientation and density of ligands like antibodies on nanomaterials of interest. Herein, the novel strategy for synthesis of a fluorescent single-walled carbon nanotube (SWNT)/tandem antibody conjugate for cellular detection of HER2 will be presented. In SWNT/tandem antibody conjugate, SWNT exhibits intrinsic near-infrared fluorescence (900-1400 nm) and Raman scattering for signal transduction of target recognition. The orientation and density of tandem antibody recognizing HER2 on SWNT are able to be effectively controlled, which results in the enhanced affinity and selectivity of a SWNT/antibody conjugate to the target molecule. The SWNT/tandem antibody conjugate was successfully applied to cellular detection of HER2 in living cells.
The past few decades have witnessed an explosion of interest in developing effective strategies for targeted drug delivery because it holds the potential to significantly enhance therapeutic efficacy while minimizing toxicity and other side effects. However, achieving specific delivery of drugs into target cells is extraordinarily challenging because the targeted drug delivery platform must simultaneously fulfill multiple requirements: it must be biocompatible with prolonged circulation, and penetrate the vasculature and other tissues to reach the target cells. Furthermore, it must internalize into the target cells and selectively release the therapeutic agent. Finally, its synthesis must be reproducible and cost effective for manufacturing. Although there are many promising approaches in the literature, they can only partially meet these requirements [Z. Cheng et al., Science (2012); A. Mullard, Nature Reviews Drug Discovery (2013)], and thus there is an urgent need for a general platform that can meet all of these criteria simultaneously.Toward this end, we describe the aptamer-polymer hybrid (APH) - a synthetic, nanoscale drug delivery vehicle (~10 nm in diameter) that specifically binds and internalizes into the target cells and selectively releases the chemotherapeutic agent (doxorubicin) for efficient delivery into the cytoplasm. To reproducibly synthesize the APH, we developed a click-chemistry scheme with a tricarboxylate ligand, and quantitatively coupled a DNA aptamer to an ethylene glycol-based block copolymer, on which multiple doxorubicin molecules were conjugated through enzymatically cleavable ester linkages. The resultant APH selectively binds to the surface marker of the target cell while the therapeutic agents remain sequestered in an inactive state. Upon internalization, the ester linkages are selectively cleaved by the esterase enzymes in the endosome, which activates the release of the doxorubicin into cytoplasm. As a proof of concept, we demonstrate an APH that specifically target breast cancer cells through the nucleolin surface marker and its selective killing of target cells. Our synthetic strategy is a general methodology; by the choice of individual components, APHs capable of targeting other cells and/or carrying different drugs in response to various stimuli will be readily synthesized in a highly controlled and modular manner.
We report the first, real-time biomechanical measurement of a DNA bundle degradation in solution when exposed to a therapeutic radiation beam. The Silicon Nano Tweezers and their microfluidic housing endure the harsh environment of radiation beams and still retain molecular-level accuracy. This result paves the way for both fundamental and clinical studies of DNA degradation under radiation for improved cancer treatment. Tumor cell killing by γ-ray beams in cancer radiotherapy is currently based on a rather empirical understanding of the basic mechanisms and effectiveness of DNA damage by radiation. On the other hand, the mechanical behavior of DNA, e.g., sequence-sensitivity, elastic vs. elastic response, is well understood. However, manipulations are usually performed by AFM or optical tweezers, instruments that can hardly be placed and operate under radiation beams.The Silicon Nano Tweezers (SNT) is a MEMS device for direct manipulation of biomolecules, an excellent candidate for in-beam operation thanks to its tiny size. The SNT comprise two parallel arms ending with sharp tips, designed to trap molecules by dielectrophoresis. The mobile arm is displaced by an electrostatic actuator. The motion is acquired by a position sensor, thus the mechanical characteristic of the trapped molecules (stiffness, viscosity) are measured in real time. SNT&’s tips are placed inside a microfluidic cavity; the alignment and the insertion are controlled by a micro-robot.The experiments are performed with a Cyberknife, a LINAC accelerator mounted on a robot arm, at the Department of Radiation Therapy of Centre Oscar Lambret. The SNT inside a microfluidic cavity is placed under the Cyberknife. The collimated beam, delivering an intense 6 MeV photon flux, completely encompasses the SNT holding the DNA bundle in the microfluidic cavity.We are working on a combined multiscale simulation scheme, ranging from molecular dynamics to Monte Carlo to continuum mechanics, which will ultimately enable to correlate the macroscopic response to the underlying molecular damage.Silicon Nanotweezers operation under therapeutic irradiation and direct detection of DNA damage under γ-ray beam was first demonstrated. Coupled with microfluidics, and in the future with biological assay technology, this new capability permits to study the mechanics of DNA damage under ionizing beams for optimized and patient-specific cancer treatments.
We developed nanoconjugation technique to allow controlled formulations of sub-100 nm, mono-modal polymeric and silica nanoconjugates with defined drug loading, quantitative drug loading efficiency and controlled release profiles. We explored the formulation as well as cancer targeting in vitro and in vivo, and demonstrated the size dependency of tumor tissue penetration and antitumor efficacy of the nanoconjugates. In a separate study, we developed chain shattering polymeric therapeutics with controlled structure and composition. Unlike conventional drug conjugates that drug release is subject to hydrolysis or enzymatic degradation of the linkage connecting the drug to the nanostructures, chain shattering polymeric therapeutics can release up to 90% of the incorporated drugs within 10 minutes in response to external triggers.
Rational combination therapies for solid tumors often exploit complex and dynamic cell signaling networks where cellular co-localization of drug compounds with disparate physiochemical properties is required for therapeutic synergy. Such control can be difficult to achieve using traditional formulations, given that individual drugs often exhibit widely varying molecular weight, charge, and lipophilicity, arriving at the tumor site at differing times and partitioning over vastly different areas. Although the delivery of multiple, distinct nanoscale drug carriers can improve tissue disposition and kinetics, vehicle-associated toxicity is compounded, limiting the safety and therapeutic index of these strategies. Self-assembled layer-by-layer (LbL) nanoparticles provide a means with which to safely and effectively deliver a range of multicomponent therapeutics to solid tumors. Recent work in the Hammond Lab has focused on the development of nanoscale LbL architectures that are both pH-responsive and active tumor-targeting. Here, we found that polyamine/glycosaminoglycan LbL architectures can achieve both low non-specific uptake and hypoxia-induced cellular delivery. These drug carriers selectively bound cell-surface CD44 receptor, a well-characterized marker for breast carcinoma cells with stem-like, mesenchymal, tumor-initiating, hypoxia-inducible, and drug-resistant phenotype. These LbL nanoparticles diminished triple-negative breast cancer cell migration and invasion, in vitro, in a receptor-selective manner. In vivo, these particles co-localized with CD44 receptor in murine tumor xenograft models, displayed ca. two-fold lower liver accumulation versus comparable anionic polysaccharide-terminal architectures, and elicited low and transient changes in plasma cytokine levels following intravenous administration. Layer-by-Layer polymer nanotechnologies provide a robust, modular, and scalable platform for multicomponent drug delivery to solid tumors.
Silica nanoparticles are interesting candidates for diagnostic as well as therapeutic agents in cancer research. Indeed first clinical trials have now been performed with such probes in melanoma patients. These trials are moving towards diagnostic applications only, however. Here we will present materials that pave the way to theranostics, i.e. a combination of diagnostic and therapeutic applications. We report a one-pot, room-temperature synthetic method for a class of mesoporous silica nanoparticles (MSNs) containing both cubic and hexagonally structured compartments within one particle. These multicompartment MSNs (mc-MSNs) consist of a core with cage-like cubic mesoporous morphology and up to four branches with hexagonally packed cylindrical mesopores epitaxially growing out of the cubic core vertices. The control parameter for the extent of branch growth from the core is a single additive concentration in the synthesis, which generates a dynamic change of synthesis condition during particle growth triggering the switch of morphologies. We discuss the results in the context of achieving high levels of architectural complexity and control in locally amorphous, mesoscopically well-defined nanoparticles for specific chemistries potentially useful in cancer theranostics.
Two-photon fluorescence materials have attracted much recent attention for their many prom-ising applications, especially in the growing field of biomedical imaging. Among the best performing two-photon fluorescence materials are semiconductor quantum dots such as CdSe and related core-shell nanoparticles. At the same time, however, heavy metals as the essential elements in available high-performance semiconductor quantum dots have prompted serious health and environmental concerns. Therefore, the search for benign alternatives has become increasingly important. Inorganic Janus particles have promised high versatility for biomedi-cal applications because of their multifunctionality. Monodisperse multifunctional and non-toxic Au@MnO nano-hetero-particles with different sizes and morphologies were prepared by a seed-mediated nucleation and growth technique with precise control over domain sizes, surface functionalization, and dye labeling. The metal oxide domain could be coated selec-tively with a thin silica layer leaving the metal domain untouched. Both, size and morphology of the individual (metal and metal oxide) domains could be controlled by adjustment of the synthetic parameters. The SiO2 coating of the oxide domain allows biomolecule conjugation (e.g. antibodies, proteins) in a single step for converting the photoluminescent and magnetic Janus nanoparticles into multifunctional efficient vehicles for theragnostics. The Au@MnO@SiO2 heterodimers were characterized using high-resolution transmission elec-tron microscopy (HR-) TEM, powder x-ray diffraction (PXRD), optical (UV-vis) spectrosco-py, confocal laser scanning microscopy (CLSM) and dynamic light scattering (DLS). The functionalized nanoparticles were stable in buffer solution or serum showing no indication of aggregation. Biocompatibility and potential biomedical applications of the Au@MnO@SiO2 Janus particles were assayed by a cell viability analysis by co-incubating the Au@MnO@ SiO2 Janus particles with Caki 1 and HeLa cells. Time-resolved fluorescence spectroscopy in combination with CLSM revealed the silica-coated Au@MnO@SiO2 heterodimers to be high-ly two-photon active; no indication for an electronic interaction between the dye molecules incorporated in the silica shell surrounding the MnO domains and the attached Au domains was found; fluorescence quenching was observed when dye molecules were bound directly to the Au domains.Dye functionalized nanoparticles were utilized for imaging throat cancer cells that overex-press the receptor for the epidermal growth factor (EGFR) on their surface. The cells can be targeted with nanoparticles carrying the corresponding antibody and detected microscopically based on their two-photon fluorescence and MRI signal in an in vivo study.
Many drugs are poorly water soluble; this limits their bioavailability and therefore their effectiveness as medication. The bioavailability increases with increasing solubility and dissolution rate of drugs. The solubility is significantly higher for amorphous compared to crystalline substances. Furthermore, the dissolution rate scales with the surface to volume ratio of drug particles. Thus, it would be highly desirable to formulate hydrophobic drugs as amorphous nanoparticles. However, techniques that allow a continuous, reliable production of amorphous sub-100 nm active ingredient particles have yet to be established. We present a poly(dimethylsiloxane) (PDMS)-based microfluidic spray dryer, a so-called microfluidic nebulator, that enables the production of amorphous sub-30 nm nanoparticles.
Cancer is a leading cause of death in the United States, in part, because the disease is usually diagnosed after it has metastasized to distant tissues and organs, at which time treatment options are limited and usually ineffective. Technologies to detect cancer at an early stage, when it is curable by current therapeutics, would provide significant benefit to cancer disease patients. Clinical measurement of biomarkers offers the promise of a noninvasive and cost effective screening for early detection of cancer. We have developed a novel 3-dimensional "nanocavity” array for the detection of human cancer biomarkers in serum and other fluids. This all-electronic diagnostic sensor is based on a nanoscale coaxial array architecture (Rizal, et al. 2013) that we have modified to enable molecular-level detection and identification. Each individual sensor in the array is a vertically-oriented coaxial capacitor, whose electrical capacitance and dielectric impedance are measurably changed when target molecules enter the coax annulus. We are designing a nanocoaxial biosensor based on electronic response to antibody recognition of a specific cancer biomarker (e.g. CA-125 for early-stage ovarian cancer) on biofunctionalized metals surfaces within the nanocoax structure, thereby providing an all-electronic, ambient temperature, rapid-response, label-free redox biosensor. Our results demonstrate the feasibility of using this array as an ultrasensitive device to detect a wide range of proteins of interest, including disease biomarkers. B. Rizal, B. Ye, P. Dhakal, T. C. Chiles, S. Shepard, G. McMahon, M. J. Burns, M. J. Naughton, Nano-optics for enhancing light-matter interactions on a molecular scale : plasmonics, photonic materials and sub-wavelength resolution, B. Di Bartolo, J. Collins, L. Silvestri (eds), Springer, Dordrecht 2013, p. 359.Supported by NIH (National Cancer Institute and the National Institute of Allergy and Infectious Diseases).
While cell migration in 3D environments has now become a standard, lack of robust models continue to hamper our ability to investigate tumor level behavior and Epithelial-to-Mesenchymal Transition (EMT) in 3D environments. This limitation significantly affects our ability to understand the signaling cascades and mechano-chemical regulators of metastasis and cancer progression. Using an integrated strategy using cell derived and naturally occurring matrices, we have developed millimeter sized tumoroids of highly invasive breast and bone cancer and have investigated the response of these tumors to external mechanical and chemical perturbation, as well as therapeutic interventions. Our method allows us to create a biomimetic environment to investigate cellular level signaling upon surgery, incision and chemotherapy. Our results with MCF-7 and U2OS cells demonstrate the fidelity and robustness of our approach and demonstrate how matrix mechanics, in conjunction with porosity and availability of adhesion ligands regulates tumoroid size, shape and EMT. Additionally, we note that matrix environment plays a strong role in mediating cellular response to both bolus and nanoparticle based chemotherapeutics. Overall, our approach represents a new strategy in creating stable, scalable and organized tumoroids and have demonstrated how matrix properties regulates tumor progression and response to therapy in native like 3D environments.
Tumor invasion and metastasis are ultimately responsible for over 90% of cancer-related fatalities, but remain poorly understood. One signature of malignant tumor progression is the detachment and dissemination of individually invading tumor cells from a collectively invading front. These complex and emergent behaviors are thought to occur as a consequence of the epithelial-mesenchymal transition (EMT), which converts adherent epithelial cells to a motile mesenchymal phenotype. Here, I integrate engineered microenvironments with high-content imaging to comprehensively measure heterogeneous invasion dynamics in populations of tens of thousands of cells. Based on their observed behaviors, cells can be clustered into distinct subpopulations that display individual or collective invasion. Remarkably, individually invading cells are associated with faster velocities as well as straighter trajectories, enabling efficient scattering and dispersal. These spatial and temporal dynamics are then physically modeled based on the solidification of binary alloys, suggesting that the governing mechanisms are differences in motility as well as phenotypic interconversion. Finally, these behaviors are perturbed using migration-inhibiting compounds, revealing that cells with different invasion phenotypes also exhibit pronounced differences in drug sensitivity.
Embryonic development consists of a complex series of cell signaling, cell migration and cell differentiation processes that are coordinated during morphogenesis. Collective cell sheet migration is an important process that sculpts the shape of an organism and its internal tissues during early development. The fate of the tissues and their ultimate physiological function relies on integrating genetic programs and processing external cues to provide positional information to guide cell migration and induce cell differentiation. By contrast, embryonic development and tissue-self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types. Considering the important role cell mechanics plays in tissue self-assembly it is surprising that little is known about the mechanical response of the multi-layer tissues to chemical cues. One of the reasons of this knowledge gap is the lack of the technologies to analyze the individual responses of epithelial and mesenchymal cell sheets in a multi cell layer tissue to mechanical cues. To investigate the processes that guide collective movements of multiple cell layers our group has focused on developing a novel microfluidic technique capable of producing complex patterns of laminar multicellular structures. We call this technique "3D tissue-etching” by analogy with the techniques used in the MEMS field. We use tissue etching to shape a complex multi-layered embryonic tissue and explore the dynamic collective responses of epithelial and mesenchymal cells in a single tissue. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents over Animal Cap tissues of Xenopus laevis. Microfluidics provides the ability to control chemical stimulation of biological systems using laminar flow patterning. To deliver precise chemical stimulation to a multicellular tissue, we have developed a system for controlling the inlet pressures to a microfluidic device by modulating fluidic resistance and capacitance. Using microfluidics, we were able to construct in a controlled multi-step etching process a complex tissue. Using etching we produce free-edges of epithelial cells over mesenchymal cells and free-edges of mesenchymal cells. We followed the process of etching and the resulting tissues with time-lapse microscopy to understand the effects of tissue etching on the mechanics of the tissue microenvironment and the impact of these changes on collective cell migration. This allows us to study multi-layer coordination of epithelial and mesenchymal cell layers and acute mechanical and behavioral response of intact epithelial and mesenchymal cell sheets to removal of neighboring or overlying tissues. The ability to control the form of multicellular tissues potentially will have a high impact in tissue engineering and regeneration applications in bioengineering and medicine
Traditional methods of quantifying cell movement in response to a chemotactic factors provide either a binary count of cell migration in response to a known concentration of the factor of interest in solution, as in Boyden chamber assays, or a method of tracking cells to determine velocities across a solubilized protein gradient where exact concentrations vary over time and are difficult to define, as in the Ibidi chemotaxis gradient assay. Using a silane self-assembling monolayer (SAM)-based procedure pioneered by V Hlady, et al. we have developed an assay capable of covalently binding a wide variety of proteins to an optically transparent surface in a 2D pattern via amine linkages. This new assay provides greater control of protein concentration and gradient intensity than possible in assays using solubilized proteins.To achieve a smooth and continuous gradient, a monolayer of beta-mercatopropyltrimethoxysilane was deposited unto a quartz substrate in an ultra-high purity Nitrogen-filled glovebox (>1.9 mm Hg) under anoxic anhydrous conditions. The silane polymerization reaction was performed by immersing an acid etched quartz slide in 99.9% anhydrous toluene and catalyzed with the addition of 5 uL of water to 100 mL of toluene. Polar/non-polar emulsion was achieved by addition of .51 mg of sodium dodecyl sulfate /mL water and sonication. Chemical gradients were formed by selectively inducing the oxidation of surface thiol groups present in the silane monolayer via UV irradiation in the presence of atmospheric oxygen, using a mask and translating stage. This process results in conversion from thiol to sulfonate groups. Protein bonding is achieved through the use of a heterobifunctional crosslinker (SMPEG12) that contains maleamide and NHS groups capable of binding selectively to thiol and amine groups respectively.Results from preliminary investigations into the silane monolayer chemistry using contact angle goniometry and X-ray photoelectron spectroscopy (XPS) demonstrate that a monolayer of silane is formed, and for specific irradiation times, there is a linear relationship between the total irradiance and conversion from thiol to sulfonate groups. Specific protein and crosslinker concentration in units of ng/um2 will be verified by XPS. Initial cell microscopy studies planned include time lapse phase contrast and fixed focal contact immunostaining (vinculin, actin, etc.) of chondrocytes and mesenchymal stem cells.
Perhaps one of the most remarkable and synthetically desirable properties of some biological cellular systems is the process known as chemotaxis. Chemotaxis is the ubiquitous biological process by which a cell is able to detect the concentration gradient of a particular chemical signal in an environment and then moves accordingly to that gradient. Bacterial E.Coli exhibit “run and tumble” chemotactic behavior in order to move towards regions that are rich in nutrients or to avoid regions of high toxicity. Here we have designed functional assemblies that can mimic biological systems that undergo chemotaxis and are sensitive to both temporal and spatial gradients. The microswimers or walkers we have designed are unique in that our walker&’s propulsion mechanism is essentially a hybrid design incorporating both biological propulsion mechanisms, such as cilia and flagella, and molecular motors, which transport cargo inside cells along the cytoskeleton. This duality enables the locomotion of the walkers themselves while still propelling the surrounding fluid, which has unique advantages for micro-scale manipulation through mimicked chemotaxis. This novel approach was realized by utilizing functionalized superparamagnetic particles or beads that spontaneously self-assemble due to magnetic forces and form chains. A rotating magnetic field drives the chains to turn and walk across the surface. The streptavidin coated particles are coupled to a surface with a gradient in the density of biotin binding sites and the velocity of the particle is modulated as it walks across the surface. As the particles encounter more binding sites the chain tends to “hinge” and translate more than it would in an area with a low density of binding sites where it tends to “slip” and translation decreases. Thus by controlling the surface-bead interactions, we can control the velocity of the walkers and detect inhomogeneous patterns on surface, effectively creating chemotactic walkers.
A key issue in the use of nanomaterials is controlling how they interact with themselves and with the outer world. Our research program focuses on the tailoring of nanoparticles of surfaces for a variety of applications, coupling the atomic-level control provided by organic synthesis with the fundamental principles of supramolecular chemistry. Using these engineered nanoparticles, we are developing particles for cancer therapeutics and diagnostics. This talk will focus on the interfacing of nanoparticles with biosystems, and will discuss our use of nanoparticles for delivery applications including our studies of small molecule, nucleic acid, and protein delivery. This presentation will also feature the use of nanoparticles for diagnostic applications, including the use of array-based sensing paradigms for the detection and identification of cancer.
Nanoparticles have emerged as a promising tool in the biomedical field, where they serve as delivery carriers of imaging agents or drugs for nanomedicines. After intravenous injection, nanoparticles show higher accumulation in angiogenic disease sites than normal tissues due to the enhanced permeability and retention (EPR) effect. To further improve their specificity to disease sites, active targeting strategies have been developed using biological targeting moieties such as antibodies, aptamers, or peptides which bind proper receptors on the surface of target cells. Efficient binding of nanoparticles to the cell surface can increase their accumulation at the target site, while unbound nanoparticles are cleared from the tissue and eliminated from the body. However, saturation of these receptors limits the capacity of targeted nanoparticles. In addition, targetable receptors are rarely unique to the disease, so nanoparticles can accumulate in other healthy tissues via these receptors resulting in reduced therapeutic efficacy and unintended side effects. Herein, we introduce a new in vivo active targeting strategy for nanoparticles via metabolic glycoengineering and bioorthogonal copper-free click chemistry in the living body. This technique is expected to enable dose-dependent and temporal generation of chemical groups on the target tumor site just like biological receptors, controlling the tumor-targetability of nanoparticles.
Targeting nanoparticles (NPs) to interact with over-expressed surface receptors on cancer cells, commonly known as active targeting, has shown potential in increasing selective cellular uptake, diminishing systematic toxicity, and enhancing therapeutic efficacy. These factors in combination have the capacity to reform cancer treatment; but thus far success of targeted drug delivery systems has been limited. There are a number of variables (e.g. nanoparticle composition, targeting ligand type, targeting ligand density, etc.) that contribute to in vivo behavior which may lead to a diminished therapeutic effect if not optimized. In this account, we studied the effects of targeting ligand density on high aspect ratio polymeric hydrogel nanoparticles. Utilizing the nanoparticle fabrication technique PRINT (Particle Replication in Non-wetting Templates), 80 nm x 80 nm x 320 nm PEG-based hydrogels were synthesized with surface amine substituent groups for post-fabrication functionalization. A FITC-tagged ZEGFR binding domain affibody was conjugated to the nanoparticle at various loadings via poly(ethylene glycol) cross-linker. Standard plate-reader analysis was used to determine ligand density, ranging from ~1000 to ~100 ligands per NP. In vitro studies in multiple cell lines indicated that particle internalization was dependent on time, NP dose concentration, and targeting ligand density. Pharmacokinetic analysis of EGFR-targeted particles indicted a strong relationship between ligand density, tumor accumulation, and circulation half-life. Overall, we have described how ligand density affects key parameters in the active targeting of NP drug delivery systems. In future work we will incorporate chemotherapeutics into our targeted nanoparticle system in order to determine therapeutic efficacy in a number of cancer models.
We herein present a miniaturized holographic imaging system for high-throughput detection and molecular profiling of rare cancer cells. In this portable imaging system, the sample is illuminated by a partially coherent light source and in-line holograms are recorded by a digital sensor array. A new computational algorithm is developed to reconstruct original images and count target cells. To detect and profile cancer cells, we first label blood samples with molecular-specific microbeads. Such labeling enables 1) a reliable differentiation between cancer cells and host cells (e.g., leukocytes); and 2) quantitative profiling of target marker expression through bead-counting. The bead-labeled blood sample is then flowed through a fluidic channel placed directly on the digital sensor array, and holographic images of cells are recorded. From these holographic images, the computational algorithm recovers the original cellular images. This reconstruction algorithm is able to retrieve both the intensity and the phase information of the original image. It also recognizes and counts the number of target cells and microbeads. By counting the number of beads attached on cells, we could measure the expression levels of different cancer markers. The profiling results agree well with that of flow cytometry and fluorescence microscopy. For real-time image reconstruction and cell counting, we have implemented parallel computing algorithm that utilizes multicore graphics processing unit (GPU). The resulting imaging system enables high throughput cellular detection - up to 1000 cells/µL could be imaged over a wide detection area (20 mm2), and cellular images could be reconstructed in real time (20 frames/sec). Furthermore, the assay could be performed without any dilution or washing steps, minimizing cell loss and simplifying the assay procedure. Cost-effective, portable, and easy-to-use, this imaging system could be readily applied in clinical settings for cancer diagnosis.
The development of both normal tissues and tumors depends on concerted interactions between cells and their local microenvironment. In the case of a tumor, this microenvironment includes not only the tumor-associated chemical and mechanical factors, but also the surrounding host cells. Here we used tissue engineering strategies to mimic these aspects of the tumor microenvironment in order to parse the relative roles of each in tumor development. We used a 3D microfabrication-based approach to normal epithelial ducts containing individual tumor cells and tumor aggregates, and microfluidic strategies to control the mechanical properties of the tissue. We found that tumor cell phenotype depended entirely on these parameters, regardless of underlying genetic abnormalities. Tumor cell invasion depended on the host mechanical stress, as shown both computationally and experimentally, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining micro-computed tomographic analysis with finite element modeling confirmed that regions of high mechanical stress correspond with regions of tumor formation in vivo. This work suggests that the mechanical tone of the non-tumorigenic host epithelium directs the phenotype of tumor cells, and provides additional insight into the instructive role of the mechanical tumor microenvironment. Our study also suggests that tissue engineering strategies can be used to unveil novel aspects of tumor development.
Cancer stem cells (CSCs) are a small fraction of cancer cells in the tumor bulk that drive the tumor initiation, progression and recurrence. CSCs, also called tumor-initiating cells, are characterized by properties including capabilities of self-renew, sphere forming, drug resistance, and tumor forming in vivo. Therefore a better understanding of the regulation of CSCs will facilitate the development of therapeutic CSC-targeting strategy for the successful eradication of cancer. However, isolated CSCs based on cell surface marker spontaneously differentiate into non-stem cancer cells in traditional two dimensional culture condition with tissue culture plastics. In our study, we have demonstrated that synthetic fibrous scaffolds generated by electrospinning can mimic the extracellular matrix in structure and provide three dimensional culture condition. A variety of cancer cell lines cultured in fibrous scaffolds significantly increased the properties of CSCs, which indicates the CSCs in those cell lines expanded in the three dimensional scaffolds. These results suggest electrospun fibrous scaffolds itself without protein components can enrich the CSCs in tumor cell lines. These fibrous scaffolds have a great potential as an in vitro platform to screen anti-CSCs drugs, model the tumor growth, as well as study the CSCs dynamics and the cell-matrix interaction.
Hydrogels are widely employed as cell culture platforms to study cellular phenomena in a 3D tissue-mimetic context. Engineered hydrogel networks have been designed to aid in understanding the influence of biophysical properties on cell behavior. In order to understand more complex cellular phenomena that take place in wound healing, disease, and development, hydrogels need to be engineered to mimic dynamic changes that occur in the microenvironment. For example, in mammary tumor progression, the extracellular matrix progressively stiffens from 100 Pa to over 1000 Pa. There is evidence that abnormally stiff matrices lead to loss of epithelial character in mammary epithelial cells and an increase in invasion. It is hypothesized that the dynamic changes in extracellular matrix properties can contribute to disease progression. Thus, it is our objective to develop a 3D dynamic hydrogel culture platform that allows for modulation of gel stiffness over time.Our approach utilizes three essential components; i) alginate, an anionic biopolymer that progressively crosslinks in the presence of divalent cations, ii) temperature-sensitive liposomes encapsulating calcium, and iii) gold nanorods that locally heat when irradiated near the absorption peak in the near-infrared region. Thus, irradiation of the gold nanorods induces a release of calcium that crosslinks the alginate hydrogel. We have demonstrated that the calcium loaded liposomes can be mixed with alginate solution and gelled upon irradiation. When distributed within a weak 3D gel and irradiated, the stiffness can be linearly increased from 91 Pa to 1548 Pa. The extent of stiffening is a function of irradiation time, which allows for temporal modulation to intermediate values to mimic gradual tissue stiffening. Alginate crosslinks are reversible, thus chelators such as citrate can be loaded in the liposomes to temporally soften gels. Using this strategy, we generated a decrease in stiffness from 1104 Pa to 467 Pa. 3T3 cells seeded within a gel consisting of alginate, calcium loaded liposomes, and Matrigel as a cell-adhesive moiety were irradiated for 30, 60 or 120 s or left unirradiated as controls. No loss of viability due to irradiation was observed through either MTS or Live/Dead assays compared to controls. Cells in the unirradiated gels adopted elongated morphologies while cells in the irradiated samples became more rounded with increasing irradiation time (thus, increasing stiffness). Finally, we have demonstrated the ability to induce gelation transdermally in a mouse model due to the high penetration depth of NIR light in tissue. We have developed a novel light-triggered system for temporal modulation of hydrogel stiffness over a physiologically relevant range. Ultimately, we intend to use this system as a tool to explore the influence of dynamic microenvironments on cell phenotype in a highly controlled and directed manner.
Despite significant progress in the development of new chemotherapeutic agents and drug delivery methods for brain tumors, malignant gliomas (high grade brain tumor) remains deadly with a median survival period of only about a year. The high dosage of chemotherapeutic agents required for penetration through the blood brain barrier during chemotherapy not only kills cancer cells but also damages healthy tissues and causes adverse side effects. Hence, a major unmet challenge in the treatment of malignant gliomas is the development of effective and targeted local delivery of chemotherapeutic agents at the cellular level. Drug-loaded biodegradable microcapsules with high drug encapsulation efficiency and controlled shape and size are attractive candidates for a more precise control of anticancer agent delivery at the tumor sites. Here, we report the results of a systematic study of the size, shape, and drug release profiles of Poly(lactic-co glycolic) (PLGA) microcapsules produced and loaded with the anticancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) using an electrojetting technique. We report production of BCNU-loaded PLGA microcapsules in the form of flattened microspheres, microspheres, and microfibers with significantly (1) higher drug encapsulation efficiency, (2) more tunable drug loading capacity, and (3) narrower size distribution than those generated using other encapsulation methods. To prepare BCNU-PLGA solutions with PLGA concentrations ranging from 1 to 10 wt%, BCNU/PLGA mixtures (5 to 25 wt% with respect to PLGA) dissolved in chloroform were electrojetted on gold substrates. We quantified the shape and size distribution of BCNU-loaded PLGA microcapsules as a function of the polymer concentration and flow rate used during electrojetting, and characterized drug release profiles for microcapsules of three different morphologies: flattened microspheres, microspheres, and microfibers. Flattened microspheres were produced from 1 and 2 wt% PLGA solutions, while microspheres and microfibers were formed as the PLGA concentration increased to 3-5 wt% and 10% wt%, respectively. We investigated the effect of flow rate (i.e. 0.25, 0.5 and 1 mLh-1) on the size and monodispersity of BCNU-loaded PLGA microcapsules. Microcapsules of 1 wt% PLGA, 4 wt% PLGA, and 10 wt% PLGA formed at a flow rate of 0.25 mLh-1 had narrower size distributions (CV = 4.6%, CV = 4.5%, and CV = 4.8%, respectively). A commonly accepted definition of monodispersity is CV < 5%. We also measured drug release profiles and developed mathematical models to estimate the effective diffusion coefficient of BCNU in different PLGA microcapsules. The BCNU release profiles for all three microcapsule morphologies were found to be in good agreement with model predictions for drug release as a result of drug diffusion and degradation of PLGA microcapsules.
Hydroxyapatite (HA)-containing microcalcifications (MCs) are routinely used for detection of breast cancer via mammography and associated with malignant disease. However, the role of MCs in the etiology of osteolytic bone metastasis, the leading cause of deaths in breast cancer patients, remains unclear. We have previously developed a polylactide-co-glycolide (PLG)-based scaffold system containing commercially available HA nanoparticles to evaluate MDA-MB231 breast cancer cell behavior in response to HA. Our results using this system suggest that interactions with HA promote the expression of bone-metastatic genes in breast cancer cells and regulate their secretion of the pro-inflammatory and osteolytic cytokine interleukin-8 (IL-8). However, to what extent these changes are related to specific HA materials properties (e.g., particle size, morphology, and crystallinity) remains unclear given the large heterogeneity of HA in commercial preparations. To overcome this limitation and analyze HA-breast cancer cell interactions as a function of varied materials properties at high-throughput, we have developed a biomimetic approach based on HA-forming simulated body fluid (SBF). More specifically, we have incubated PLG coated 96-well plates with SBF, a solution containing inorganic components of human blood plasma, and have analyzed the resulting coatings by SEM, FT-IR, and XRD to assess morphology, composition and phase of coatings, respectively. Our results suggest that SBF carbonate concentration is inversely correlated with the crystallinity of the HA surfaces formed. Furthermore, MDA-MB231 cultured on variations of these mineralized surfaces up-regulated IL-8 secretion in response to increased HA crystallinity, while exhibiting decreased adhesive and proliferative capacity. These results are corroborated with similar findings from PLG scaffolds incorporated with synthetic HA of high and low crystallinities, which validates the relevance of our approach. Taken together, these data suggest that biomimetic mineral-coatings are an effective tool with which precisely defined cell-mineral interactions may be efficiently assessed.
Two major promising applications of nanotechnology towards the treatment of cancer are the control over molecular binding sites and the detection of binding phenomena, which could lead to better therapies and earlier detection. Recent advances in the ability to control matter at the nanoscale have enabled unprecedented capabilities using semiconductor and polymeric materials. Bringing together expertise in nanoscience, chemistry, and biology, our laboratory is developing these two classes of materials both alone and in combination to control molecular recognition. This work has a wide range of potential therapeutic and diagnostic applications. One focus of our lab is the development of fluorescent single-walled carbon nanotubes as optical sensors to create monitors for cancer biomarkers. These materials allow the detection of binding events down to the single-molecule level, in real-time, and in a minimally-invasive way. Encapsulation of nanotubes in synthetic polymers and biopolymers creates a handle for the transduction of analyte binding. Small molecules, such as reactive oxygen species and other genotoxins, can be detected by transduction of the binding event to the polymer or the nanotube itself. Multiplexed analyte identification is possible by observing variations in the nanotube&’s spectral response, resulting in distinct optical fingerprints. Nanotube emission can undergo both wavelength and intensity modulation, permitting the identification of analytes which are difficult to differentiate via conventional methods. The analyte responses can be spatially mapped in live cells and tissues, measured with sensitivity down to the single-molecule level, and detected in real-time, facilitating new and unprecedented biological measurements.
We have recently developed a miniaturized microfluidic chip-based technology, the micro-Hall detector (uHD), that can perform rapid, highly sensitive, and quantitative measurement of individual cells in unprocessed biological samples. The uHD detects the Hall voltage induced by magnetic moments of cells in-flow that have been magnetically tagged with magnetic nanoparticles (MNPs) and bio-orthogonal chemistry. The entire assay is performed on a single microfluidic chip without the need for washing and purification steps, thereby allowing cellular diagnostics to be conducted in point-of-care settings. As a proof-of-concept, we previously implemented the first uHD prototype that had 8 micro-Hall elements. The system was used to detect MNP-labeled Gram-positive bacteria. The detection limit was similar to that of culture tests (~10 bacteria), but with a 50-times faster assay time. The application was further extended to detect and profile circulating tumor cells (CTCs) in the whole blood of cancer patients.To improve the assay throughput and sensitivity, we designed the next generation uHD for practical applications. The new system incorporates a larger array of micro-Hall elements within a wide fluidic channel to increase sensing area, thus flowing higher sample volume. Signal processing circuits are integrated in the same chip, which helps to maximize signal-to-noise ratio (SNR). With such capabilities, the new uHD could be a comprehensive and universal diagnostic platform with potential for broad applications in resource-limited, point-of-care settings.
Surface plasmon resonance (SPR) biosensors have been widely used to study the interactions of proteins, small molecules, and nucleotides. Their sensing range (50 ~ 200 nm) parallels the size of exosomes, which are nano-sized membrane-bound vesicles released from cells. Here, we demonstrate a label-free and sensitive approach for molecular profiling of exosomes using a novel SPR nanohole platform. The device comprises arrays of nanoholes, patterned onto a thin gold film on a glass substrate. With a multi-channel flow cell constructed on top of the sensor arrays, each nanohole array was functionalized with a different type of antibody. Target-specific binding of exosomes on the array induced spectral shifts in the resonance wavelength. Such shifts were proportional to the number of bound exosomes and correlate sensitively with overall cancer antigen abundance, therefore enabling quantitative molecular profiling. The nanoholes displayed high detection sensitivity; using CD63 as a pan-exosomal marker, we established a detection threshold of ~ 2,000 exosomes. We also profiled the expression level of molecular markers (e.g., EpCAM, HER2, EGFR) in exosomes isolated from cancer cell culture, whose profiling results showed an excellent match with those of parental cells. This indicates that cancer-derived exosomes reflect the molecular signature of their primary tumor. With its capacity for sensitive, label-free molecular detection, the plasmonic nanoholes would be a promising new platform for comprehensive exosomal molecular analyses.