Optimizing Oxygen-Production Kinetics of Manganese Dioxide Nanoparticles Improves Hypoxia Reversal and Survival in Mice with Bone Metastases.

Persistent hypoxia in bone metastases induces an immunosuppressive environment, limiting the effectiveness of immunotherapies. To address chronic hypoxia, we have developed manganese dioxide (MnO2) nanoparticles with tunable oxygen production kinetics for sustained oxygenation in bone metastases lesions. Using polyethylene glycol (PEG)-stabilized MnO2 or poly(lactic[50]-co-glycolic[50] acid) (50:50 PLGA), poly(lactic[75]-co-glycolic[25] acid) (75:25 PLGA), and polylactic acid (PLA)-encapsulated MnO2 NPs, we demonstrate that polymer hydrophobicity attenuates burst oxygen production and enables tunable oxygen production kinetics. The PEG-MnO2 NPs resulted in rapid hypoxia reduction in spheroids, which was rapidly attenuated, while the PLA-MnO2 NPs exhibited delayed hypoxia control in cancer spheroids. The 50:50 PLGA-MnO2 NPs exhibited the best short- and long-term control of hypoxia in cancer spheroids, resulting in sustained regulation of the expression of HIF-1α and immunosuppressive genes. The sustained control of hypoxia by the 50:50 PLGA-MnO2 NPs enhanced the cytotoxicity of natural killer cells against cancer spheroids. In vivo, 50:50 PLGA-MnO2 showed greater accumulation in the long bones and pelvis, common sites for bone metastases. The NPs decreased hypoxia in bone metastases and decreased regulatory T cell levels, resulting in enhanced survival of mice with established bone metastases.

Vascular bifurcation influences the protein corona composition on nanoparticles and impacts their cellular uptake.

The protein corona (PC) that forms on nanoparticles (NPs) after in vivo injection influences their biodistribution, pharmacokinetics, and cell interaction. Although injected NPs traverse vascular networks, the impact of vascular features on the protein corona composition is mainly unexplored. Using an in vitro flow model that introduces bifurcations, a common feature of blood vessels, we show that vessels are not passive bystanders in the formation of the PC but that their features play active roles in defining the PC on NPs. The addition of bifurcation significantly increased the amount of proteins associated with NP. The bifurcation's introduction also changed the PC's composition on the NPs and affected the NP interactions with cells. Correlation analysis and modeling showed that these changes in the PC are mediated by both the branching and diameter reduction associated with vessel bifurcation and the resulting change in flow rate. The results indicate that blood vessel structures play an active part in the information of the PC, and their role should be studied critically for a better understanding of the PC and its biological implications.

Reversing Hypoxia with PLGA-Encapsulated Manganese Dioxide Nanoparticles Improves Natural Killer Cell Response to Tumor Spheroids.

The adoptive transfer of natural killer (NK) cells, which can recognize and obliterate cancer cells, provides a practical alternative to current treatment modalities to improve cancer patients' survival. However, translating NK cell therapies to treat solid tumors has proven challenging due to the tumor microenvironment (TME). Hypoxia in the TME induces immunosuppression that inhibits the cytotoxic function of NK cells. Thus, reversing hypoxia-induced immunosuppression is critical for effective adoptive NK cell immunotherapy. In this study, we use manganese dioxide nanoparticles (MnO2 NPs) to catalyze the degradation of tumor-produced hydrogen peroxide, thereby generating oxygen. For improved biocompatibility and modulation of oxygen production, the MnO2 NPs were encapsulated into poly(lactic-co-glycolic) to produce particles that are 116 nm in size and with a ζ-potential of +17 mV (PLGA-MnO2 NPs). The PLGA-MnO2 NPs showed first-order oxygen production and sustained high oxygen tension compared to equivalent amounts of bare MnO2 NPs in the presence of H2O2. The PLGA-MnO2 NPs were biocompatible, reduced hypoxia after penetration into the core of cancer spheroids, and decreased hypoxia-induced factor 1 α expression. Reducing hypoxia in the spheroid resulted in a decrease in the potent immunosuppressors, adenosine, and lactate, which was confirmed by electrospray ionization mass spectroscopy (ESI-MS). ESI-MS also showed a change in the metabolism of the amino acids aspartate, glutamine, and glutamate after hypoxia reduction in the cancer cells. Notably, the spheroids' microenvironment changes enhanced NK cells' cytotoxicity, which obliterated the spheroids. These results demonstrate that reducing hypoxia-induced immunosuppression in tumors is a potent strategy to increase the potency of cytotoxic immune cells in the TME. The developed NPs are promising new tools to improve adoptive NK cell therapy.

Engineered Three-Dimensional Tumor Models to Study Natural Killer Cell Suppression.

A critical hurdle associated with natural killer (NK) cell immunotherapies is inadequate infiltration and function in the solid tumor microenvironment. Well-controlled 3D culture systems could advance our understanding of the role of various biophysical and biochemical cues that impact NK cell migration in solid tumors. The objectives of this study were to establish a biomaterial which (i) supports NK cell migration and (ii) recapitulates features of the in vivo solid tumor microenvironment, to study NK infiltration and function in a 3D system. Using peptide-functionalized poly(ethylene glycol)-based hydrogels, the extent of NK-92 cell migration was observed to be largely dependent on the density of integrin binding sites and the presence of matrix metalloproteinase degradable sites. When lung cancer cells were encapsulated into the hydrogels to create tumor microenvironments, the extent of NK-92 cell migration and functional activity was dependent on the cancer cell type and duration of 3D culture. NK-92 cells showed greater migration into the models consisting of nonmetastatic A549 cells relative to metastatic H1299 cells, and reduced migration in both models when cancer cells were cultured for 7 days versus 1 day. In addition, the production of NK cell-related pro-inflammatory cytokines and chemokines was reduced in H1299 models relative to A549 models. These differences in NK-92 cell migration and cytokine/chemokine production corresponded to differences in the production of various immunomodulatory molecules by the different cancer cells, namely, the H1299 models showed increased stress ligand shedding and immunosuppressive cytokine production, particularly TGF-ß. Indeed, inhibition of TGF-ß receptor I in NK-92 cells restored their infiltration in H1299 models to levels similar to that in A549 models and increased overall infiltration in both models. Relative to conventional 2D cocultures, NK-92 cell mediated cytotoxicity was reduced in the 3D tumor models, suggesting the hydrogel serves to mimic some features of the biophysical barriers in in vivo tumor microenvironments. This study demonstrates the feasibility of a synthetic hydrogel system for investigating the biophysical and biochemical cues impacting NK cell infiltration and NK cell-cancer cell interactions in the solid tumor microenvironment.

Manganese dioxide nanoparticles protect cartilage from inflammation-induced oxidative stress.

Oxidative stress has been implicated in the pathogenesis of osteoarthritis and has become an important therapeutic target. Investigations of various antioxidant supplements, reactive oxidative species (ROS) pathway mediators, and free radical scavengers for treating osteoarthritis have demonstrated common disadvantages including poor bioavailability and stability, as well as rapid joint clearance or release profiles from delivery vehicles. Moreover, these therapies do not target cartilage, which irreversibly degenerates in the presence of oxidative stress. The goal of this study was to engineer a nanoparticle system capable of sustained retention in the joint space, localization to cartilage, and mitigation of oxidative stress. Towards this goal, ROS scavenging manganese dioxide nanoparticles with physicochemical properties (less than 20 nm and cationic) that facilitate their uptake into cartilage were developed and characterized. These particles penetrated through the depth of cartilage explants and were found both in the extracellular matrix as well as intracellularly within the resident chondrocytes. Furthermore, the particles demonstrated chondroprotection of cytokine-challenged cartilage explants by reducing the loss of glycosaminoglycans and release of nitric oxide. Quantitative PCR analysis revealed that the particles mitigated impacts of oxidative stress related genes in cytokine-challenged chondrocytes. When injected intra-articularly into rats, the particles persisted in the joint space over one week, with 75% of the initial signal remaining in the joint. Biodistribution and histological analysis revealed accumulation of particles at the chondral surfaces and colocalization of the particles with the lacunae of chondrocytes. The results suggest that the manganese dioxide nanoparticles could be a promising approach for the chondroprotection of osteoarthritic cartilage.

Multifunctional nanoparticles for intracellular drug delivery and photoacoustic imaging of mesenchymal stem cells.

Strategies that control the differentiation of mesenchymal stem cells (MSC) and enable image-guided cell implantation and longitudinal monitoring could advance MSC-based therapies for bone defects and injuries. Here we demonstrate a multifunctional nanoparticle system that delivers resveratrol (RESV) intracellularly to improve osteogenesis and enables photoacoustic imaging of MSCs. RESV-loaded nanoparticles (RESV-NPs), formulated from poly (lactic-co-glycolic) acid and iron oxide, enhanced the stability of RESV by 18-fold and served as photoacoustic tomography (PAT) contrast for MSCs. Pre-loading MSCs with RESV-NP upregulated RUNX2 expression with a resultant increase in mineralization by 27% and 45% compared to supplementation with RESV-NP and free RESV, respectively, in 2-dimensional cultures. When grown in polyethylene glycol-based hydrogels, MSCs pre-loaded with RESV-NPs increased the overall level and homogeneity of mineralization compared to those supplemented with free RESV or RESV-NP. The PAT detected RESV-NP-loaded MSCs with a resolution of 1500 cells/µL, which ensured imaging of MSCs upon encapsulation in a PEG-based hydrogel and implantation within the rodent cranium. Significantly, RESV-NP-loaded MSCs in hydrogels did not show PAT signal dilution over time or a reduction in signal upon osteogenic differentiation. This multifunctional NP platform has the potential to advance translation of stem cell-based therapies, by improving stem cell function and consistency via intracellular drug delivery, and enabling the use of a promising emerging technology to monitor cells in a clinically relevant manner.

Nanoparticle Properties for Delivery to Cartilage: The Implications of Disease State, Synovial Fluid, and Off-Target Uptake.

A major hurdle limiting the ability to treat and cure osteoarthritis, a common and debilitating disease, is rapid joint clearance and limited cartilage targeting of intra-articular therapies. Nanoscale drug carriers have the potential to improve therapeutic targeting and retention in the joint after direct injection; however, there still lacks a fundamental understanding of how the physicochemical properties of nanoparticles (NPs) influence localization to the degenerating cartilage and how joint conditions such as disease state and synovial fluid impact NP biodistribution. The goal of this study was to assess how physicochemical properties of NPs influence their interactions with joint tissues and, ultimately, cartilage localization. Ex vivo models of joint tissues were used to study how poly(lactide- co-glycolide) (PLGA) and polystyrene (PS) NP size, charge, and surface chemistry influence cartilage retention under normal and disease-mimicking conditions. Of the particles investigated, PLGA NPs surface-modified with a quaternary ammonium cation had the greatest retention within cartilage explants; however, retention was diminished 2- to 2.9-fold in arthritic tissue and in the presence of synovial fluid. Interactions with synovial fluid induced changes to NP surface properties and colloidal stability in vitro. The impact of NP charge on "off-target" synoviocyte uptake was also dependent on synovial fluid interactions. The results suggest that the design of nanocarriers for targeted drug delivery within the joint cannot be based on a single parameter such as zeta potential or size, and that the fate of injected delivery systems will likely be influenced by the disease state of the joint and the presence of synovial fluid.

Targeted Nanomedicine to Treat Bone Metastasis.

Bone metastases are common complications of solid tumors, particularly those of the prostate, breast, and lungs. Bone metastases can lead to painful and devastating skeletal-related events (SREs), such as pathological fractures and nerve compressions. Despite advances in treatment for cancers in general, options for bone metastases remain inadequate and generally palliative. Anticancer drugs (chemotherapy and radiopharmaceuticals) do not achieve therapeutic concentrations in the bone and are associated with dose-limiting side effects to healthy tissues. Nanomedicines, with their tunable characteristics, have the potential to improve drug targeting to bone metastases while decreasing side effects for their effective treatment. In this review, we present the current state of the art for nanomedicines to treat bone metastases. We also discuss new treatment modalities enhanced by nanomedicine and their effects on SREs and disease progression.

Inhibition of bone loss with surface-modulated, drug-loaded nanoparticles in an intraosseous model of prostate cancer.

Advanced-stage prostate cancer usually metastasizes to bone and is untreatable due to poor biodistribution of intravenously administered anticancer drugs to bone. In this study, we modulated the surface charge/composition of biodegradable nanoparticles (NPs) to sustain their blood circulation time and made them small enough to extravasate through the openings of the bone's sinusoidal capillaries and thus localize into marrow. NPs with a neutral surface charge, achieved by modulating the NP surface-associated emulsifier composition, were more effective at localizing to bone marrow than NPs with a cationic or anionic surface charge. These small neutral NPs (~150nm vs. the more usual ~320nm) were also ~7-fold more effective in localizing in bone marrow than large NPs. We hypothesized that NPs that effectively localize to marrow could improve NP-mediated anticancer drug delivery to sites of bone metastasis, thereby inhibiting cancer progression and preventing bone loss. In a PC-3M-luc cell-induced osteolytic intraosseous model of prostate cancer, these small neutral NPs demonstrated greater accumulation in bone within metastatic sites than in normal contralateral bone as well as co-localization with the tumor mass in marrow. Significantly, a single-dose intravenous administration of these small neutral NPs loaded with paclitaxel (PTX-NPs), but not anionic PTX-NPs, slowed the progression of bone metastasis. In addition, neutral PTX-NPs prevented bone loss, whereas animals treated with the rapid-release drug formulation Cremophor EL (PTX-CrEL) or saline (control) showed >50% bone loss. Neutral PTX-NPs did not cause acute toxicity, whereas animals treated with PTX-CrEL experienced weight loss. These results indicate that NPs with appropriate physical and sustained drug-release characteristics could be explored to treat bone metastasis, a significant clinical issue in prostate and other cancers.

Advancements in the delivery of epigenetic drugs.

INTRODUCTION: Advancements in epigenetic treatments are not only coming from new drugs, but also from modifications or encapsulation of the existing drugs into different formulations leading to greater stability and enhanced delivery to the target site. The epigenome is highly regulated and complex; therefore, it is important that off-target effects of epigenetic drugs be minimized. The step from in vitro to in vivo treatment of these drugs often requires development of a method of effective delivery for clinical translation. AREAS COVERED: This review covers epigenetic mechanisms such as DNA methylation, chromatin remodeling and small-RNA-mediated gene regulation. There is a section in the review with examples of diseases where epigenetic alterations lead to impaired pathways, with an emphasis on cancer. Epigenetic drugs, their targets and clinical status are presented. Advantages of using a delivery method for epigenetic drugs as well as examples of current advancements and challenges are also discussed. EXPERT OPINION: Epigenetic drugs have the potential to be very effective therapy against a number of diseases, especially cancers and neurological disorders. As with many chemotherapeutics, undesired side effects need to be minimized. Finding a suitable delivery method means reducing side effects and achieving a higher therapeutic index. Each drug may require a unique delivery method exploiting the drug's chemistry or other physical characteristic requiring interdisciplinary participation and would benefit from a better understanding of the mechanisms of action.

Modulation of the tumor microenvironment for cancer treatment: a biomaterials approach.

Tumors are complex tissues that consist of stromal cells, such as fibroblasts, immune cells and mesenchymal stem cells, as well as non-cellular components, in addition to neoplastic cells. Increasingly, there is evidence to suggest that these non-neoplastic cell components support cancer initiation, progression and metastasis and that their ablation or reprogramming can inhibit tumor growth. Our understanding of the activities of different parts of the tumor stroma in advancing cancer has been improved by the use of scaffold and matrix-based 3D systems originally developed for regenerative medicine. Additionally, drug delivery systems made from synthetic and natural biomaterials deliver drugs to kill stromal cells or reprogram the microenvironment for tumor inhibition. In this article, we review the impact of 3D tumor models in increasing our understanding of tumorigenesis. We also discuss how different drug delivery systems aid in the reprogramming of tumor stroma for cancer treatment.

Nanoparticles: cellular uptake and cytotoxicity.

Understanding the interactions of nanoparticles (NPs) with cells and how these interactions influence their cellular uptake is essential to exploring the biomedical applications of NPs, particularly for drug delivery. Various factors, whether differences in physical properties of NPs or variations in cell-membrane characteristics, influence NP-cell interactions and uptake processes. NP-cell membrane interactions may also influence intracellular trafficking of NPs, their sorting into different intracellular compartments, cellular retention, and hence the efficacy of encapsulated therapeutics. A crucial consideration is whether such interactions might cause any toxicity, starting with how NPs interact in transit with the biological environment prior to their interactions with targeted cells and tissues. Understanding the effects of various NP characteristics on cellular and biological processes could help in designing NPs that are efficient but also nontoxic.

Heterogeneity in nanoparticles influences biodistribution and targeting.

AIM: A large fraction of the administered dose of nanoparticles (NPs) localizes into nontarget tissue, which could be due to the heterogeneous population of NPs. MATERIALS & METHODS: To investigate the impact of the above issue, we simultaneously tracked the biodistribution using optical imaging of two different sized poly(d,l-lactide co-glycolide) NPs, which also varied in their surface charge and texture, in a prostate tumor xenograft mouse model. RESULTS: Although formulated using the same polymer and emulsifier concentration, small NPs were neutral (S-neutral-NPs), whereas large NPs were anionic (L-anionic-NPs). Simultaneous injection of these NPs, representing heterogeneity, shows significantly different biodistribution. S-neutral-NPs demonstrated longer circulation time than L-anionic-NPs (t1/2 = 96 vs 13 min); accounted for 75% of total NPs accumulated in the tumor; and showed 13-fold greater tumor to liver signal intensity ratio than L-anionic-NPs. CONCLUSION: The data underscore the importance of formulating nanocarriers of specific properties to enhance their targeting efficacy.

Optical imaging to map blood-brain barrier leakage.

Vascular leakage in the brain is a major complication associated with brain injuries and certain pathological conditions due to disruption of the blood-brain barrier (BBB). We have developed an optical imaging method, based on excitation and emission spectra of Evans Blue dye, that is >1000-fold more sensitive than conventional ultraviolet spectrophotometry. We used a rat thromboembolic stroke model to validate the usefulness of our method for vascular leakage. Optical imaging data show that vascular leakage varies in different areas of the post-stroke brain and that administering tissue plasminogen activator causes further leakage. The new method is quantitative, simple to use, requires no tissue processing, and can map the degree of vascular leakage in different brain locations. The high sensitivity of our method could potentially provide new opportunities to study BBB leakage in different pathological conditions and to test the efficacy of various therapeutic strategies to protect the BBB.

Selective biophysical interactions of surface modified nanoparticles with cancer cell lipids improve tumor targeting and gene therapy.

Targeting gene- or drug-loaded nanoparticles (NPs) to tumors and ensuring their intratumoral retention after systemic administration remain key challenges to improving the efficacy of NP-based therapeutics. Here, we investigate a novel targeting approach that exploits changes in lipid metabolism and cell membrane biophysics that occur during malignancy. We hypothesized that modifications to the surface of NPs that preferentially increase their biophysical interaction with the membrane lipids of cancer cells will improve intratumoral retention and in vivo efficacy upon delivery of NPs loaded with a therapeutic gene. We have demonstrated that different surfactants, incorporated onto the NPs' surface, affect the biophysical interactions of NPs with the lipids of cancer cells and normal endothelial cells. NPs surface modified with didodecyldimethylammoniumbromide (DMAB) demonstrated greater interaction with cancer cell lipids, which was 6.7-fold greater than with unmodified NPs and 5.5-fold greater than with endothelial cell lipids. This correlated with increased uptake of DMAB-modified NPs with incubation time by cancer cells compared to other formulations of NPs and to uptake by endothelial cells. Upon systemic injection, DMAB-NPs demonstrated a 4.6-fold increase in tumor accumulation compared to unmodified NPs which also correlated to improved efficacy of p53 gene therapy. Characterization of the biophysical interactions between NPs and lipid membranes of tumors or other diseased tissues/organs may hold promise for engineering targeted delivery of therapeutics.