Design and Evaluation of Nanoscale Materials with Programmed Responsivity towards Epigenetic Enzymes.

Self-assembled materials capable of modulating their assembly properties in response to specific enzymes play a pivotal role in advancing 'intelligent' encapsulation platforms for biotechnological applications. Here, we introduce a previously unreported class of synthetic nanomaterials that programmatically interact with histone deacetylase (HDAC) as the triggering stimulus for disassembly. These nanomaterials consist of co-polypeptides comprising poly (acetyl L-lysine) and poly(ethylene glycol) blocks. Under neutral pH conditions, they self-assemble into particles. However, their stability is compromised upon exposure to HDACs, depending on enzyme concentration and exposure time. Our investigation, utilizing HDAC8 as the model enzyme, revealed that the primary mechanism behind disassembly involves a decrease in amphiphilicity within the block copolymer due to the deacetylation of lysine residues within the particles' hydrophobic domains. To elucidate the response mechanism, we encapsulated a fluorescent dye within these nanoparticles. Upon incubation with HDAC, the nanoparticle structure collapsed, leading to controlled release of the dye over time. Notably, this release was not triggered by denatured HDAC8, other proteolytic enzymes like trypsin, or the co-presence of HDAC8 and its inhibitor. We further demonstrated the biocompatibility and cellular effects of these materials and conducted a comprehensive computational study to unveil the possible interaction mechanism between enzymes and particles. By drawing parallels to the mechanism of naturally occurring histone proteins, this research represents a pioneering step toward developing functional materials capable of harnessing the activity of epigenetic enzymes such as HDACs.

An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvß3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.

In Vitro Tumor Mimetic Spheroid Model: Void Space within a Self-Detachable Cross-Linked Hydrogel.

The three-dimensional (3D) spheroid cell culture model is crucial in screening anticancer drugs in vitro and understanding tumor cell behavior. However, the current in vitro models require highly skilled techniques. Here, we present an in vitro, tumor-mimetic, self-detachable, cancer cell spheroid model that provides the confined space of a tumor microenvironment, convenient spheroid retrieval, immunostaining, treatment, and imaging. We formed a void space within alginate macrobeads by ionic disintegration at a specific region inside. The macrobeads were further destabilized with bovine serum albumin to retrieve the spheroid cultured within the void space. Quantitative analysis of the immunofluorescence images of the cultured spheroids showed enhanced expressions of the hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase-9 (CA-9), like monolayer cultures of cancer cells under hypoxic conditions (0.2% oxygen). Furthermore, adding CoCl2 to the cell culture media induces even higher amounts of HIF-1α and CA-9 in the cultured spheroids. In conclusion, the present work highlighted the in vitro spheroid model, which is closer to the tumor microenvironment and has user-friendly cell seeding, spheroid retrieval, and immunostaining steps.

Material Properties, Dissolution and Time Evolution of PEGylated Lipid-Shelled Microbubbles: Effects of the Polyethylene Glycol Hydrophilic Chain Configurations.

Polyethylene glycol (PEG) is often added to the lipid coating of a contrast microbubble to prevent coalescence and improve circulation. At high surface density, PEG chains are known to undergo a transition from a mushroom configuration to an extended brush configuration. We investigated the effects of PEG chain configuration on attenuation and dissolution of microbubbles by varying the molar ratio of the PEGylated lipid in the shell with three (0%, 2% and 5%) in the mushroom configuration and two (10% and 20%) in the brush configuration. We measured attenuation through the bubble suspensions and used it to obtain the characteristic rheological properties of their shells according to two interfacial rheological models. The interfacial elasticity was found to be significantly lower in the brush regime (∼0.6 N/m) than in the mushroom regime (∼1.3 N/m), but similar in value within each regime. The dissolution behavior of microbubbles under acoustic excitation inside an air-saturated medium was studied by measuring the time-dependent attenuation. Total attenuation recorded a transient increase because of growth resulting from air influx and an eventual decrease caused by dissolution. Microbubble shell composition with varying PEG concentrations had significant effects on dissolution dynamics.

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvß3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 µM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells.

Integrin-targeting arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

Targeting Estrogen Receptor-Positive Breast Microtumors with Endoxifen-Conjugated, Hypoxia-Sensitive Polymersomes.

Endoxifen is the primary active metabolite of tamoxifen, a nonsteroidal-selective estrogen receptor modulator (SERM) and widely used medication to treat estrogen receptor-positive (ER+) breast cancer. In this study, endoxifen was conjugated to the surface of polymeric nanoparticles (polymersomes) for targeted delivery of doxorubicin (DOX) to estrogen receptor-positive breast cancer cells (MCF7). Rapid cell growth and insufficient blood supply result in low oxygen concentration (hypoxia) within the solid breast tumors. The polymersomes developed here are prepared from amphiphilic copolymers of polylactic acid (PLA) and poly(ethylene glycol) (PEG) containing diazobenzene as the hypoxia-responsive linker. We prepared two nanoparticle formulations: DOX-encapsulated hypoxia-responsive polymersomes (DOX-HRPs) and endoxifen-conjugated, DOX-encapsulated hypoxia-responsive polymersomes (END-DOX-HRPs). Cellular internalization studies demonstrated eight times higher cytosolic and nuclear localization after incubating breast cancer cells with END-DOX-HRPs (targeted polymersomes) in contrast to DOX-HRPs (nontargeted polymersomes). Cytotoxicity studies on monolayer cell cultures exhibited that END-DOX-HRPs were three times more toxic to ER+ MCF7 cells than DOX-HRPs and free DOX in hypoxia. The cell viability studies on three-dimensional hypoxic cultures also demonstrated twice as much toxicity when the spheroids were treated with targeted polymersomes instead of nontargeted counterparts. This is the first report of surface-decorated polymeric nanoparticles with endoxifen ligands for targeted drug delivery to ER+ breast cancer microtumors. The newly designed endoxifen-conjugated, hypoxia-responsive polymersomes might have translational potential for ER+ breast cancer treatment.

Polymeric Composite Matrix with High Biobased Content as Pharmaceutically Relevant Molecular Encapsulation and Release Platform.

Drug delivery systems (DDS) that can temporally control the rate and extent of release of therapeutically active molecules find applications in many clinical settings, ranging from infection control to cancer therapy. With an aim to design a locally implantable, controlled-release DDS, we demonstrated the feasibility of using cellulose nanocrystal (CNC)-reinforced poly (l-lactic acid) (PLA) composite beads. The performance of the platform was evaluated using doxorubicin (DOX) as a model drug for applications in triple-negative breast cancer. A facile, nonsolvent-induced phase separation (NIPS) method was adopted to form composite beads. We observed that CNC loading within these beads played a critical role in the mechanical stability, porosity, water uptake, diffusion, release, and pharmacological activity of the drug from the delivery system. When loaded with DOX, composite beads significantly controlled the release of the drug in a pH-dependent pattern. For example, PLA/CNC beads containing 37.5 wt % of CNCs showed a biphasic release of DOX, where 41 and 82% of the loaded drug were released at pH 7.4 and pH 5.5, respectively, over 7 days. Drug release followed Korsmeyer's kinetics, indicating that the release mechanism was mostly diffusion and swelling-controlled. We showed that DOX released from drug-loaded PLA/CNC composite beads locally suppressed the growth and proliferation of triple-negative breast cancer cells, MBA-MB-231, via the apoptotic pathway. The efficacy of the DDS was evaluated in human tissue explants. We envision that such systems will find applications for designing biobased platforms with programmed stability and drug delivery functions.

Dynamic cellular biomechanics in responses to chemotherapeutic drug in hypoxia probed by atomic force spectroscopy.

The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12-24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55-75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

Targeted polymeric nanoparticles for drug delivery to hypoxic, triple-negative breast tumors.

High recurrence and metastasis to vital organs are the major characteristics of triple-negative breast cancer (TNBC). Low vascular oxygen tension promotes resistance to chemo- and radiation therapy. Neuropilin-1 (NRP-1) receptor is highly expressed on TNBC cells. The tumor-penetrating iRGD peptide interacts with the NRP-1 receptor, triggers endocytosis and transcytosis, and facilitates penetration. Herein, we synthesized a hypoxia-responsive diblock PLA-diazobenzene-PEG copolymer and prepared self-assembled hypoxia-responsive polymersomes (Ps) in an aqueous buffer. The iRGD peptide was incorporated into the polymersome structure to make hypoxia-responsive iRGD-conjugated polymersomes (iPs). Doxorubicin (DOX) was encapsulated in the polymersomes to prepare both targeted and non-targeted hypoxia-responsive polymersomes (DOX-iPs and DOX-Ps, respectively). The polymeric nanoparticles released less than 30% of their encapsulated DOX within 12 hours under normoxic conditions (21% oxygen), whereas under hypoxia (2% Oxygen), doxorubicin release remarkably increased to over 95%. The targeted polymersomes significantly decreased TNBC cells' viability in monolayer and spheroid cultures under hypoxia compared to normoxia. Animal studies displayed that targeted polymersomes significantly diminished tumor growth in xenograft nude mice. Overall, the targeted polymersomes exhibited potent anti-tumor activity in monolayer, spheroid, and animal models of TNBC. With further developments, the targeted nanocarriers discussed here might have the translational potential as drug carriers for the treatment of TNBC.

Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria.

The tumor microenvironment is characterized by anomalous vascularization, hypoxia, and acidity at the core of solid tumors that culminates in concentrated necrosis and immune system dysregulation among other effects. While this environment presents several challenges for the development of oncotherapeutics that deliver their activity via the enhanced permeability and retention (EPR) effect of the leaky blood vessels around a tumor, oncolytic bacteria, or a class of bacteria with a noted capacity to lyse solid tumors, are attracted to the very environment found at the center of solid tumors that confounds other therapeutics. It is this capacity that allows for a potent, active penetration from the tumor margins into the core, and subsequent colonization to facilitate lysis and immune reactivation. Clostridium novyi in particular has recently shown great promise in preclinical and clinical trials when administered directly to the tumor. These studies indicate that C. novyi is uniquely poised to effectively accomplish the long sought after "holy grail" of oncotherapeutics: selective tumor localization via intravenous delivery. This study reports the development of efficient methods that facilitate experimental work and therapeutic translation of C. novyi including the ability to work with this obligate micro-anaerobe on the benchtop. Additionally, this study seeks to utilize this newfound experimental flexibility to address several gaps in the current knowledge regarding the efficacy of CRIPSR/Cas9-mediated gene insertion in this species to further develop this oncolytic bacteria and the genetic customization of bacteria in general.

pH-Sensitive Nanodrug Carriers for Codelivery of ERK Inhibitor and Gemcitabine Enhance the Inhibition of Tumor Growth in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), a metabolic disorder, remains one of the leading cancer mortality sources worldwide. An initial response to treatments, such as gemcitabine (GEM), is often followed by emergent resistance reflecting an urgent need for alternate therapies. The PDAC resistance to GEM could be due to ERK1/2 activity. However, successful ERKi therapy is hindered due to low ligand efficiency, poor drug delivery, and toxicity. In this study, to overcome these limitations, we have designed pH-responsive nanoparticles (pHNPs) with a size range of 100-150 nm for the simultaneous delivery of ERKi (SCH 772984) and GEM with tolerable doses. These pHNPs are polyethylene glycol (PEG)-containing amphiphilic polycarbonate block copolymers with tertiary amine side chains. They are systemically stable and capable of improving in vitro and in vivo drug delivery at the cellular environment's acidic pH. The functional analysis indicates that the nanomolar doses of ERKi or GEM significantly decreased the 50% growth inhibition (IC50) of PDAC cells when encapsulated in pHNPs compared to free drugs. The combination of ERKi with GEM displayed a synergistic inhibitory effect. Unexpectedly, we uncover that the minimum effective dose of ERKi significantly promotes GEM activities on PDAC cells. Furthermore, we found that pHNP-encapsulated combination therapy of ERKi with GEM was superior to unencapsulated combination drug therapy. Our findings, thus, reveal a simple, yet efficient, drug delivery approach to overcome the limitations of ERKi for clinical applications and present a new model of sensitization of GEM by ERKi with no or minimal toxicity.

Chemical Architecture of Block Copolymers Differentially Abrogate Cardiotoxicity and Maintain the Anticancer Efficacy of Doxorubicin.

The molecular architecture of pH-responsive amphiphilic block copolymers, their self-assembly behavior to form nanoparticles (NPs), and doxorubicin (DOX)-loading technique govern the extent of DOX-induced cardiotoxicity. We observed that the choice of pH-sensitive tertiary amines, surface charge, and DOX-loading techniques within the self-assembled NPs strongly influence the release and stimulation of DOX-induced cardiotoxicity in primary cardiomyocytes. However, covalent conjugation of DOX to a pH-sensitive nanocarrier through a "conditionally unstable amide" linkage (PCPY-cDOX; PC = polycarbonate and PY = 2-pyrrolidine-1-yl-ethyl-amine) significantly reduced the cardiotoxicity of DOX in cardiomyocytes as compared to noncovalently encapsulated DOX NPs (PCPY-eDOX). When these formulations were tested for drug release in serum-containing media, the PCPY-cDOX systems showed prolonged control over drug release (for ∼72 h) at acidic pH compared to DOX-encapsulated nanocarriers, as expected. We found that DOX-encapsulated nanoformulations triggered cardiotoxicity in primary cardiomyocytes more acutely, while conjugated systems such as PCPY-cDOX prevented cardiotoxicity by disabling the nuclear entry of the drug. Using 2D and 3D (spheroid) cultures of an ER + breast cancer cell line (MCF-7) and a triple-negative breast cancer cell line (MDA-MB-231), we unravel that, similar to encapsulated systems (PCPY-eDOX-type) as reported earlier, the PCPY-cDOX system suppresses cellular proliferation in both cell lines and enhances trafficking through 3D spheroids of MDA-MB-231 cells. Collectively, our studies indicate that PCPY-cDOX is less cardiotoxic as compared to noncovalently encapsulated variants without compromising the chemotherapeutic properties of the drug. Thus, our studies suggest that the appropriate selection of the nanocarrier for DOX delivery may prove fruitful in shifting the balance between low cardiotoxicity and triggering the chemotherapeutic potency of DOX.

Hypoxia-Responsive, Polymeric Nanocarriers for Targeted Drug Delivery to Estrogen Receptor-Positive Breast Cancer Cell Spheroids.

Uncontrolled cell growth, division, and lack of enough blood supply causes low oxygen content or hypoxia in cancerous tumor microenvironments. 17ß-Estradiol (E2), an estrogen receptor (ER) ligand, can be incorporated on the surface of nanocarriers for targeted drug delivery to breast cancer cells overexpressing ER. In the present study, we synthesized estradiol-conjugated hypoxia-responsive polymeric nanoparticles (polymersomes) encapsulating the anticancer drug doxorubicin (E2-Dox-HRPs) for targeted delivery into the hypoxic niches of estrogen-receptor-positive breast cancer microtumors. Estradiol-conjugated polymersomes released over 90% of their encapsulated Dox in a sustained manner within hypoxia (2% oxygen) after 12 h. However, they released about 30% of Dox in normal oxygen partial pressure (21% oxygen, normoxia) during this time. Fluorescence microscopic studies demonstrated higher cytosolic and nuclear internalization of E2-Dox-HRPs (targeted polymersomes) compared to those of Dox-HRPs (nontargeted polymersomes). Monolayer cell viability studies on ER-positive MCF7 cells showed higher cytotoxicity of targeted polymersomes in hypoxia compared to in normoxia. Cytotoxicity studies with hypoxic three-dimensional spheroid cultures of MCF7 cells treated with targeted polymersomes indicated significant differences compared to those of normoxic spheroids. The novel estradiol-conjugated hypoxia-responsive polymersomes described here have the potential for targeted drug delivery in estrogen-receptor-positive breast cancer therapy.

Functional Applications of Polyarginine-Hyaluronic Acid-Based Electrostatic Complexes.

Background: Electrostatic complexes of poly (l-Arginine) (pArg) and hyaluronic acid (HA) have been investigated for their functional applications to supply free or polymeric form of l-Arginine (Arg) to target cells. As a vital amino acid, Arg plays significant role in multitude of pathophysiological processes ranging from wound healing to cancer. However, serum arginase expression and toxicity of Arg at cellular level renders exogenous delivery of this amino acid a challenging task. We showed that polyarginine-hyaluronic acid ionic nanocomplexes (pArg-HA iNCs) could be an effective way to deliver Arg to target cell populations. Materials and Methods: These electrostatic complexes were prepared by mixing HA (average m.w. of 200 kDa) with pArg (m.w. 5-15 kDa; Sigma) in aqueous solutions and purifying over glycerol. Nanocomplexes were characterized for their particle size, surface charge, capacity to release l-Arg, and intracellular uptake of complexes. Results: Synthesized nanocomplexes showed hydrodynamic diameter ranging from 140-306 nm depending on the content of pArg or HA within the formulation. With surface charge (ζ-potential) of -29 mV, the nanocomplexes showed pH-dependent release of Arg. At pH 7.4, pArg-HA iNCs released 30% of the total Arg-content, while at pH 5.0, 60% of Arg was released after 24 h. These electrostatically stabilized complexes were found to promote growth of human dermal fibroblasts (HDF) in wound-healing assay and increased nitric oxide (NO) activity in these cells in a time-dependent manner. Nanocomplexes also showed cellular uptake and enhanced dose-dependent toxicity against two pancreatic cancer cell lines, i.e. MIA PaCa-2 and Panc-1. Interestingly, the cytotoxic effect was synergized upon pre-treatment of the cells with a frontline chemotherapeutic agent, gemcitabine (GEM), and was not observed when the cells were treated with Arg alone. Conclusion: As such, this communication shows the prospect of pArg-HA iNC electrostatic nanocomplexes to interact and interfere with intracellular Arg metabolic machinery conducive to rescuing different pathological conditions.

Targeting the Tumor Core: Hypoxia-Responsive Nanoparticles for the Delivery of Chemotherapy to Pancreatic Tumors.

In pancreatic ductal adenocarcinoma (PDAC), early onset of hypoxia triggers remodeling of the extracellular matrix, epithelial-to-mesenchymal transition, increased cell survival, the formation of cancer stem cells, and drug resistance. Hypoxia in PDAC is also associated with the development of collagen-rich, fibrous extracellular stroma (desmoplasia), resulting in severely impaired drug penetration. To overcome these daunting challenges, we created polymer nanoparticles (polymersomes) that target and penetrate pancreatic tumors, reach the hypoxic niches, undergo rapid structural destabilization, and release the encapsulated drugs. In vitro studies indicated a high cellular uptake of the polymersomes and increased cytotoxicity of the drugs under hypoxia compared to unencapsulated drugs. The polymersomes decreased tumor growth by nearly 250% and significantly increased necrosis within the tumors by 60% in mice compared to untreated controls. We anticipate that these polymer nanoparticles possess a considerable translational potential for delivering drugs to solid hypoxic tumors.

Echogenic Exosomes as ultrasound contrast agents.

Exosomes are naturally secreted extracellular bilayer vesicles (diameter 40-130 nm), which have recently been found to play a critical role in cell-to-cell communication and biomolecule delivery. Their unique characteristics-stability, permeability, biocompatibility and low immunogenicity-have made them a prime candidate for use in delivering cancer therapeutics and other natural products. Here we present the first ever report of echogenic exosomes, which combine the benefits of the acoustic responsiveness of traditional microbubbles with the non-immunogenic and small-size morphology of exosomes. Microbubbles, although effective as ultrasound contrast agents, are restricted to intravascular usage due to their large size. In the current study, we have rendered bovine milk-derived exosomes echogenic by freeze drying them in the presence of mannitol. Ultrasound imaging and direct measurement of linear and nonlinear scattered responses were used to investigate the echogenicity and stability of the prepared exosomes. A commercial scanner registered enhancement (28.9% at 40 MHz) in the brightness of ultrasound images in presence of echogenic exosomes at 5 mg/mL. The exosomes also showed significant linear and nonlinear scattered responses-11 dB enhancement in fundamental, 8.5 dB in subharmonic and 3.5 dB in second harmonic all at 40 µg/mL concentration. Echogenic exosomes injected into the tail vein of mice and the synovial fluid of rats resulted in significantly higher brightness-as much as 300%-of the ultrasound images, showing their promise in a variety of in vivo applications. The echogenic exosomes, with their large-scale extractability from bovine milk, lack of toxicity and minimal immunogenic response, successfully served as ultrasound contrast agents in this study and offer an exciting possibility to act as an effective ultrasound responsive drug delivery system.

Overcoming Hurdles in Nanoparticle Clinical Translation: The Influence of Experimental Design and Surface Modification.

Nanoparticles are becoming an increasingly popular tool for biomedical imaging and drug delivery. While the prevalence of nanoparticle drug-delivery systems reported in the literature increases yearly, relatively little translation from the bench to the bedside has occurred. It is crucial for the scientific community to recognize this shortcoming and re-evaluate standard practices in the field, to increase clinical translatability. Currently, nanoparticle drug-delivery systems are designed to increase circulation, target disease states, enhance retention in diseased tissues, and provide targeted payload release. To manage these demands, the surface of the particle is often modified with a variety of chemical and biological moieties, including PEG, tumor targeting peptides, and environmentally responsive linkers. Regardless of the surface modifications, the nano-bio interface, which is mediated by opsonization and the protein corona, often remains problematic. While fabrication and assessment techniques for nanoparticles have seen continued advances, a thorough evaluation of the particle's interaction with the immune system has lagged behind, seemingly taking a backseat to particle characterization. This review explores current limitations in the evaluation of surface-modified nanoparticle biocompatibility and in vivo model selection, suggesting a promising standardized pathway to clinical translation.

Exosomes as Drug Carriers for Cancer Therapy.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30-120 nm in size, exosomes are secreted from cells and isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity-all critical to the success of any nanoparticle drug delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid tumors.

Microenvironment-sensing, nanocarrier-mediated delivery of combination chemotherapy for pancreatic cancer.

Limited effectiveness of Raf and MEK inhibitors has impelled the interest to use the inhibitors of Extra-cellular Receptor Kinase (ERK) pathway in combination with Gemcitabine (GEM) in pancreatic cancer. However, off-target abundance of ERK receptors, challenging physico-chemical properties, and dose-limiting toxicity of the inhibitor has presented critical challenges towards fabricating this combination amenable for clinical translation. Herein we report a pharmaceutical nanoformulation of GEM and an ERK inhibitor (SCH 772984) co-stabilized within a pH-sensing nanocarrier (NC, with a hydrodynamic diameter of 161 ± 5.0 nm). The NCs were modularly derived from a triblock, self-assembling copolymer, and were chemically conjugated with GEM and encapsulated with SCH772984 at a loading content of 20.2% and 18.3%, respectively. Through pH-mediated unfolding of the individual blocks of the copolymer, the NCs were able to control the release of encapsulated drugs, traffic through cellular membranes, engage target receptors, suppress proliferation of pancreatic cancer cells, and accumulate at disease sites. Collectively our studies showed the feasibility of co-delivery of a combination chemotherapy consisting of GEM and an ERK inhibitor from a NC platform, which can sense and respond to tumor microenvironment of pancreatic cancer setting.