Towards clinical translation of 'second-generation' regenerative stroke therapies: hydrogels as game changers?

Stroke is an unmet clinical need with a paucity of treatments, at least in part because chronic stroke pathologies are prohibitive to 'first-generation' stem cell-based therapies. Hydrogels can remodel the hostile stroke microenvironment to aid endogenous and exogenous regenerative repair processes. However, no clinical trials have yet been successfully commissioned for these 'second-generation' hydrogel-based therapies for chronic ischaemic stroke regeneration. This review recommends a path forward to improve hydrogel technology for future clinical translation for stroke. Specifically, we suggest that a better understanding of human host stroke tissue-hydrogel interactions in addition to the effects of scaling up hydrogel volume to human-sized cavities would help guide translation of these second-generation regenerative stroke therapies.

Silk Hydrogel Substrate Stress Relaxation Primes Mesenchymal Stem Cell Behavior in 2D.

Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1ß, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1ß signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels.

Focal drug administration via heparin-containing cryogel microcarriers reduces cancer growth and metastasis.

Developing drug delivery systems that release anticancer drugs in a controlled and sustained manner remains challenging. We hypothesized that highly sulfated heparin-based microcarriers would allow electrostatic drug binding and controlled release. In silico modelling showed that the anticancer drug doxorubicin has affinity for the heparin component of the microcarriers. Experimental results showed that the strong electrostatic interaction was reversible, allowing both doxorubicin loading and a subsequent slow release over 42 days without an initial burst release. The drug-loaded microcarriers were able to reduce cancer cell viability in vitro in both hormone-dependent and highly aggressive triple-negative human breast cancer cells. Focal drug treatment, of an in vivo orthotopic triple-negative breast cancer model significantly decreased tumor burden and reduced cancer metastasis, whereas systemic administration of an equivalent drug dose was ineffective. This study proves that heparin-based microcarriers can be used as drug delivery platforms, for focal delivery and sustained long-term drug release.

Nanomedicines for the Delivery of Biologics.

A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca's initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future.

PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles.

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles.

Impact of the hypoxic phenotype on the uptake and efflux of nanoparticles by human breast cancer cells.

Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells.

Reverse-engineered silk hydrogels for cell and drug delivery.

Silk is an important biopolymer for (bio)medical applications because of its unique and highly versatile structure and its robust clinical track record in human medicine. Silk can be processed into many material formats, including physically and chemically cross-linked hydrogels that have almost limitless applications ranging from tissue engineering to biomedical imaging and sensing. This concise review provides a detailed background of silk hydrogels, including silk structure-function relationships, biocompatibility and biodegradation, and it explores recent developments in silk hydrogel utilization, with specific reference to drug and cell delivery. We address common pitfalls and misconceptions while identifying emerging opportunities, including 3D printing.

Degradation Behavior of Silk Nanoparticles-Enzyme Responsiveness.

Silk nanoparticles are viewed as promising vectors for intracellular drug delivery as they can be taken up into cells by endocytosis and trafficked to lysosomes, where lysosomal enzymes and the low pH trigger payload release. However, the subsequent degradation of the silk nanoparticles themselves still requires study. Here, we report the responsiveness of native and PEGylated silk nanoparticles to degradation following exposure to proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, a cysteine protease. Both native and PEGylated silk nanoparticles showed similar degradation behavior over a 20 day exposure period (degradation rate: protease XIV > papain ≫ α-chymotrypsin). Within 1 day, the silk nanoparticles were rapidly degraded by protease XIV, resulting in a ∼50% mass loss, an increase in particle size, and a reduction in the amorphous content of the silk secondary structure. By contrast, 10 days of papain treatment was necessary to observe any significant change in nanoparticle properties, and α-chymotrypsin treatment had no effect on silk nanoparticle characteristics over the 20-day study period. Silk nanoparticles were also exposed ex vivo to mammalian lysosomal enzyme preparations to mimic the complex lysosomal microenvironment. Preliminary results indicated a 45% reduction in the silk nanoparticle size over a 5-day exposure. Overall, the results demonstrate that silk nanoparticles undergo enzymatic degradation, but the extent and kinetics are enzyme-specific.

Silk nanoparticles: proof of lysosomotropic anticancer drug delivery at single-cell resolution.

Silk nanoparticles are expected to improve chemotherapeutic drug targeting to solid tumours by exploiting tumour pathophysiology, modifying the cellular pharmacokinetics of the payload and ultimately resulting in trafficking to lysosomes and triggering drug release. However, experimental proof for lysosomotropic drug delivery by silk nanoparticles in live cells is lacking and the importance of lysosomal pH and enzymes controlling drug release is currently unknown. Here, we demonstrate, in live single human breast cancer cells, the role of the lysosomal environment in determining silk nanoparticle-mediated drug release. MCF-7 human breast cancer cells endocytosed and trafficked drug-loaded native and PEGylated silk nanoparticles (∼100 nm in diameter) to lysosomes, with subsequent drug release from the respective carriers and nuclear translocation within 5 h of dosing. A combination of low pH and enzymatic degradation facilitated drug release from the silk nanoparticles; perturbation of the acidic lysosomal pH and inhibition of serine, cysteine and threonine proteases resulted in a 42% ± 2.2% and 33% ± 3% reduction in nuclear-associated drug accumulation for native and PEGylated silk nanoparticles, respectively. Overall, this study demonstrates the importance of lysosomal activity for anticancer drug release from silk nanoparticles, thereby providing direct evidence for lysosomotropic drug delivery in live cells.

Metabolic Reprogramming of Macrophages Exposed to Silk, Poly(lactic-co-glycolic acid), and Silica Nanoparticles.

Monitoring macrophage metabolism in response to nanoparticle exposure provides new insights into biological outcomes, such as inflammation or toxicity, and supports the design of tailored nanomedicines. This paper describes the metabolic signature of macrophages exposed to nanoparticles ranging in diameter from 100 to 125 nm and made from silk, poly(lactic-co-glycolic acid) or silica. Nanoparticles of this size and type are currently at various stages of preclinical and clinical development for drug delivery applications. 1 H NMR analysis of cell extracts and culture media is used to quantify the changes in the intracellular and extracellular metabolomes of macrophages in response to nanoparticle exposure. Increased glycolytic activity, an altered tricarboxylic acid cycle, and reduced ATP generation are consistent with a proinflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system, and a few osmolytes and antioxidants emerge as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature is a common response to all nanoparticles tested; however, the direction and magnitude of some variations are clearly nanoparticle specific, indicating material-induced biological specificity. Overall, metabolic reprogramming of macrophages can be achieved with nanoparticle treatments, modulated through the choice of the material, and monitored using 1 H NMR metabolomics.

Manufacture and Drug Delivery Applications of Silk Nanoparticles.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles - a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Heparin-Modified Polyethylene Glycol Microparticle Aggregates for Focal Cancer Chemotherapy.

Focal cancer therapy can improve clinical outcomes. Here, we evaluated injectable heparin-containing hydrogel material loaded with doxorubicin as a focal breast cancer therapy. We utilized a binary heparin/polyethylene glycol (PEG) hydrogel that was processed post synthesis into hydrogel microparticle aggregates to yield a readily injectable hydrogel. When loaded with doxorubicin, the injectable hydrogel microparticle aggregates had excellent short- and long-term anticancer activity against human breast cancer cells in vitro. Efficacy as a focal anticancer therapy was also evaluated in vivo by local injection of the doxorubicin-loaded PEG-heparin hydrogel microparticle aggregates into mice with established human orthotopic breast tumors. Animals showed significant antitumor responses by reduction in both primary tumor growth and metastasis when compared to animals that received the equivalent doxorubicin dose via an intravenous bolus injection. Overall, PEG-heparin hydrogel microparticle aggregates are emerging as a potential anticancer drug delivery system for focal therapy.

PEGylated Silk Nanoparticles for Anticancer Drug Delivery.

Silk has a robust clinical track record and is emerging as a promising biopolymer for drug delivery, including its use as nanomedicine. However, silk-based nanomedicines still require further refinements for full exploitation of their potential; the application of "stealth" design principals is especially necessary to support their evolution. The aim of this study was to develop and examine the potential of PEGylated silk nanoparticles as an anticancer drug delivery system. We first generated B. mori derived silk nanoparticles by driving ß-sheet assembly (size 104 ± 1.7 nm, zeta potential -56 ± 5.6 mV) using nanoprecipitation. We then surface grafted polyethylene glycol (PEG) to the fabricated silk nanoparticles and verified the aqueous stability and morphology of the resulting PEGylated silk nanoparticles. We assessed the drug loading and release behavior of these nanoparticles using clinically established and emerging anticancer drugs. Overall, PEGylated silk nanoparticles showed high encapsulation efficiency (>93%) and a pH-dependent release over 14 days. Finally, we demonstrated significant cytotoxicity of drug loaded silk nanoparticles applied as single and combination nanomedicines to human breast cancer cells. In conclusion, these results, taken together with prior silk nanoparticle data, support a viable future for silk-based nanomedicines.

Focal therapy of neuroblastoma using silk films to deliver kinase and chemotherapeutic agents in vivo.

Current methods for treatment of high-risk neuroblastoma patients include surgical intervention, in addition to systemic chemotherapy. However, only limited therapeutic tools are available to pediatric surgeons involved in neuroblastoma care, so the development of intraoperative treatment modalities is highly desirable. This study presents a silk film library generated for focal therapy of neuroblastoma; these films were loaded with either the chemotherapeutic agent doxorubicin or the targeted drug crizotinib. Drug release kinetics from the silk films were fine-tuned by changing the amount and physical crosslinking of silk; doxorubicin loaded films were further refined by applying a gold nanocoating. Doxorubicin-loaded, physically crosslinked silk films showed the best in vitro activity and superior in vivo activity in orthotopic neuroblastoma studies when compared to the doxorubicin-equivalent dose administered intravenously. Silk films were also suitable for delivery of the targeted drug crizotinib, as crizotinib-loaded silk films showed an extended release profile and an improved response both in vitro and in vivo when compared to freely diffusible crizotinib. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based anticancer drug delivery systems.

Tissue engineering a surrogate niche for metastatic cancer cells.

In breast and prostate cancer patients, the bone marrow is a preferred site of metastasis. We hypothesized that we could use tissue-engineering strategies to lure metastasizing cancer cells to tissue-engineered bone marrow. First, we generated highly porous 3D silk scaffolds that were biocompatible and amenable to bone morphogenetic protein 2 functionalization. Control and functionalized silk scaffolds were subcutaneously implanted in mice and bone marrow development was followed. Only functionalized scaffolds developed cancellous bone and red bone marrow, which appeared as early as two weeks post-implantation and further developed over the 16-week study period. This tissue-engineered bone marrow microenvironment could be readily manipulated in situ to understand the biology of bone metastasis. To test the ability of functionalized scaffolds to serve as a surrogate niche for metastasis, human breast cancer cells were injected into the mammary fat pads of mice. The treatment of animals with scaffolds had no significant effect on primary tumor growth. However, extensive metastasis was observed in functionalized scaffolds, and the highest levels for scaffolds that were in situ manipulated with receptor activator of nuclear factor kappa-B ligand (RANKL). We also applied this tissue-engineered bone marrow model in a prostate cancer and experimental metastasis setting. In summary, we were able to use tissue-engineered bone marrow to serve as a target or "trap" for metastasizing cancer cells.

Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments.

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell-secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow-specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.

Self-assembling doxorubicin silk hydrogels for the focal treatment of primary breast cancer.

Standard care for early stage breast cancer includes tumor resection and local radiotherapy to achieve long-term remission. Systemic chemotherapy provides only low locoregional control of the disease; therefore, we describe self-assembling silk hydrogels that can retain and then deliver doxorubicin locally. Self-assembling silk hydrogels show no swelling, are readily loaded with doxorubicin under aqueous conditions and release drug over 4 weeks in amounts that can be fine-tuned by varying the silk content. Following successful in vitro studies, locally injected silk hydrogels loaded with doxorubicin show excellent antitumor response in mice, outperforming the equivalent amount of doxorubicin delivered intravenously. In addition to reducing primary tumor growth, doxorubicin-loaded silk hydrogels reduce metastatic spread and are well tolerated in vivo. Thus, silk hydrogels are well suited for the local delivery of chemotherapy and provide a promising approach to improve locoregional control of breast cancer.

pH-dependent anticancer drug release from silk nanoparticles.

Silk has traditionally been used as a suture material because of its excellent mechanical properties and biocompatibility. These properties have led to the development of different silk-based material formats for tissue engineering and regenerative medicine. Although there have been a small number of studies about the use of silk particles for drug delivery, none of these studies have assessed the potential of silk to act as a stimulus-responsive anticancer nanomedicine. This report demonstrates that an acetone precipitation of silk allows the formation of uniform silk nanoparticles (98 nm diameter, polydispersity index 0.109), with an overall negative surface charge (-33.6 ± 5.8 mV), in a single step. Silk nanoparticles are readily loaded with doxorubicin (40 ng doxorubicin/µg silk) and show pH-dependent release (pH 4.5≫ 6.0 > 7.4). In vitro studies with human breast cancer cell lines demonstrates that the silk nanoparticles are not cytotoxic (IC50 > 120 µg mL(-1) ) and that doxorubicin-loaded silk nanoparticles are able to overcome drug resistance mechanisms. Live cell fluorescence microscopy studies show endocytic uptake and lysosomal accumulation of silk nanoparticles. In summary, the pH-dependent drug release and lysosomal accumulation of silk nanoparticles demonstrate the ability of drug-loaded silk nanoparticles to serve as a lysosomotropic anticancer nanomedicine.

Stem Cell Implants for Cancer Therapy: TRAIL-Expressing Mesenchymal Stem Cells Target Cancer Cells In Situ.

PURPOSE: Tumor-specific delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing peptide, at effective doses remains challenging. Herein we demonstrate the utility of a scaffold-based delivery system for sustained therapeutic cell release that capitalizes on the tumor-homing properties of mesenchymal stem cells (MSCs) and their ability to express genetically-introduced therapeutic genes. METHODS: Implants were formed from porous, biocompatible silk scaffolds seeded with full length TRAIL-expressing MSCs (FLT-MSCs). under a doxycycline inducible promoter. In vitro studies with FLT-MSCs demonstrated TRAIL expression and antitumor effects on breast cancer cells. Next, FLT-MSCs were administered to mice using three administration routes (mammary fat pad co-injections, tail vein injections, and subcutaneous implantation on scaffolds). RESULTS: In vitro cell-specific bioluminescent imaging measured tumor cell specific growth in the presence of stromal cells and demonstrated FLT-MSC inhibition of breast cancer growth. FLT-MSC implants successfully decreased bone and lung metastasis, whereas liver metastasis decreased only with tail vein and co-injection administration routes. Average tumor burden was decreased when doxycycline was used to induce TRAIL expression for co-injection and scaffold groups, as compared to controls with no induced TRAIL expression. CONCLUSION: This implant-based therapeutic delivery system is an effective and completely novel method of anticancer therapy and holds great potential for clinical applications.