Thermosensitive Polymeric Nanoparticles for Drug Co-Encapsulation and Breast Cancer Treatment.

Despite advances in breast cancer treatment, there remains a need for local management of noninvasive, low-grade ductal carcinoma in situ (DCIS). These focal lesions are well suited for local intraductal treatment. Intraductal administration supported target site drug retention, improved efficacy, and reduced systemic exposure. Here, we used a poly(N-isopropyl acrylamide, pNIPAM) nanoparticle delivery system loaded with cytotoxic piplartine and an MAPKAP Kinase 2 inhibitor (YARA) for this purpose. For tumor environment targeting, a collagen-binding peptide SILY (RRANAALKAGELYKSILYGSG-hydrazide) was attached to pNIPAM nanoparticles, and the nanoparticle diameter, zeta potential, drug loading, and release were assessed. The system was evaluated for cytotoxicity in a 2D cell culture and 3D spheroids. In vivo efficacy was evaluated using a chemical carcinogenesis model in female Sprague-Dawley rats. Nanoparticle delivery significantly reduced the IC50 of piplartine (4.9 times) compared to the drug in solution. The combination of piplartine and YARA in nanoparticles further reduced the piplartine IC50 (~15 times). Treatment with these nanoparticles decreased the in vivo tumor incidence (5.2 times). Notably, the concentration of piplartine in mammary glands treated with nanoparticles (35.3 ± 22.4 µg/mL) was substantially higher than in plasma (0.7 ± 0.05 µg/mL), demonstrating targeted drug retention. These results indicate that our nanocarrier system effectively reduced tumor development with low systemic exposure.

Collagen-Binding Nanoparticles for Paclitaxel Encapsulation and Breast Cancer Treatment.

In this study, we developed a novel hybrid collagen-binding nanocarrier for potential intraductal administration and local breast cancer treatment. The particles were formed by the encapsulation of nanostructured lipid carriers (NLCs) containing the cytotoxic drug paclitaxel within a shell of poly(N-isopropylacrylamide) (pNIPAM), and were functionalized with SILY, a peptide that binds to collagen type I (which is overexpressed in the mammary tumor microenvironment) to improve local retention and selectivity. The encapsulation of the NLCs in the pNIPAM shell increased nanoparticle size by approximately 140 nm, and after purification, a homogeneous system of hybrid nanoparticles (∼96%) was obtained. The nanoparticles exhibited high loading efficiency (<76%) and were capable of prolonging paclitaxel release for up to 120 h. SILY-modified nanoparticles showed the ability to bind to collagen-coated surfaces and naturally elaborated collagen. Hybrid nanoparticles presented cytotoxicity up to 3.7-fold higher than pNIPAM-only nanoparticles on mammary tumor cells cultured in monolayers. In spheroids, the increase in cytotoxicity was up to 1.8-fold. Compared to lipid nanoparticles, the hybrid nanoparticle modified with SILY increased the viability of nontumor breast cells by up to 1.59-fold in a coculture model, suggesting the effectiveness and safety of the system.

Chitosan hydrogels with MK2 inhibitor peptide-loaded nanoparticles to treat atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder that lacks ideal long-term treatment options due to a series of side effects, such as skin atrophy, related to the most common treatment prescribed to manage moderate-to-severe AD. In this study, a cell-penetrating MK2 inhibitor peptide YARA (YARAAARQARAKALNRQGLVAA) was loaded into hollow thermo-responsive pNIPAM nanoparticles (NP), which were further incorporated into chitosan hydrogels (H-NP-YARA) to promote local drug delivery, improve moisture and the anti-inflammatory activity. The NPs exhibited high loading efficiency (>50%) and the hydrogel remained porous following NP incorporation as observed by scanning electron microscopy (SEM). Both nanoparticles and hydrogels were able to improve the release of YARA and sustained release to up to 120 h. The hydrogels and NPs delivered 2 and 4-fold more YARA into viable skin layers of porcine skin in vitro at 12 h post-application than the non-encapsulated compound in intact and impaired barrier conditions. Furthermore, the YARA-loaded NPs (NP-YARA) and H-NP-YARA treatment decreased the levels of inflammatory cytokines up to 20 time-fold compared with the non-treated group of human keratinocytes under inflammatory conditions. Consistent with the results in cell culture, the loading of YARA in NP reduced the levels of IL-1ß, IL-6, and TNF-α up to 3.3 times in an ex vivo skin culture model after induction of inflammation. A further decrease of up to 17 times-fold was observed with H-NP-YARA treatment compared to the drug in solution. Our data collectively suggest that chitosan hydrogel containing YARA-loaded nanoparticles is a promising new formulation for the topical treatment of AD.

A comparison of electrosprayed vs vortexed glycosaminoglycan-peptide nanoparticle platform for protection and improved delivery of therapeutic peptides.

Therapeutic peptides capable of reducing inflammation via inhibition of the MAP kinase 2 pathway have the potential to reduce inflammation in atopic dermatitis by suppressing secretion of inflammatory cytokines by resident keratinocytes. One of the biggest hurdles to the use of therapeutic peptides, however, is their rapid degradation by intrinsic proteases and peptidases found in serum. Here we introduce a new nanoparticle technology that enhances and extends the bioactivity of a MAP KAP kinase 2 inhibitor peptide (MK2i) via electrostatic complexation with Dermatan sulfate (DS), a glycosaminoglycan, and explore their properties under various conditions. DS-MK2i nanoparticles can be made using electrospray ionization or sonication and vortexing with no stabilizing polymers or crosslinking. Average particle diameter, polydispersity index, and zeta potential were measured over a pH range of 2.5-11.5, in increments of 0.5, in water and at physiological ionic strength. Both particle types were shown to be shelf stable, robust, and behave differently in response to pH. They are also significantly more effective at suppressing cytokine secretion in inflamed, human keratinocytes than peptide alone in the presence of serum, providing a facile method of protecting peptides for therapeutic delivery in conditions such as atopic dermatitis, and abrogating the need for serum-starvation in in vitro testing.

Electroconductive agarose hydrogels modulate mesenchymal stromal cell adhesion and spreading through protein adsorption.

Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.

Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair.

Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation. Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration. Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs. Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.

Thermoresponsive, hollow, degradable core-shell nanoparticles for intra-articular delivery of anti-inflammatory peptide.

Inflammation following joint trauma contributes to cartilage degradation and progression of post traumatic osteoarthritis (PTOA). Therefore, drug delivery vehicles that deliver effective anti-inflammatory treatments have the potential to prevent PTOA. We have developed solid and hollow, thermoresponsive nanoparticles for the controlled release of our anti-inflammatory MK2-inhibiting (MK2i) peptide for intra-articular injection to halt inflammation that contributes to the advancement of PTOA. This system exploits the thermosensitive characteristic of N-isopropyl acrylamide (NIPAm) to transition phases when passing through its lower critical solution temperature (LCST). The nanoparticles (NPs) swell below the LCST and constrict above it. Non-crosslinked poly(NIPAm) (pNIPAm), held above its LCST, formed hydrophobic cores around which shells composed of NIPAm, degradable crosslinker N, N'-bis (acryloyl) cystamine (BAC), sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and acrylic acid (AAc) were polymerized. Removal of the non-crosslinked pNIPAm cores via diffusion produced thermosensitive, degradable nanoparticles with low density, or hollow, cores. The data presented here revealed low-density, termed hollow, nanoparticles (hNPs) load and release significantly more MK2i than solid nanoparticles (sNPs). Furthermore, drug loading below the LCST of NIPAm results in roughly 2.5 times more therapeutic encapsulation compared to loading particles in their constricted state. Hollow nanoparticles increase drug loading compared to solid nanoparticles, are taken up into chondrocytes within 24 h, cleared from the cells within 6 days, significantly decrease the secretion of the proinflammatory cytokine IL-6, and, via intra-articular injection, are successfully delivered into the joint space of rats. The peptide loaded nanoparticles provide a reproducible platform for intra-articular delivery of therapeutics.

Multi-peptide presentation and hydrogel mechanics jointly enhance therapeutic duo-potential of entrapped stromal cells.

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Collagen Type I and II Blend Hydrogel with Autologous Mesenchymal Stem Cells as a Scaffold for Articular Cartilage Defect Repair.

Collagen type II is a promising material to repair cartilage defects since it is a major component of articular cartilage and plays a key role in chondrocyte function. This study investigated the chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) embedded within a 3:1 collagen type I to II blend (Col I/II) hydrogel or an all collagen type I (Col I) hydrogel. Glycosaminoglycan (GAG) production in Col I/II hydrogels was statistically higher than that in Col I hydrogels or pellet culture, and these results suggested that adding collagen type II promoted GAG production. Col I/II hydrogels had statistically lower alkaline phosphatase (AP) activity than pellets cultured in a chondrogenic medium. The ability of MSCs encapsulated in Col I/II hydrogels to repair cartilage defects was investigated by creating two cartilage defects in the femurs of rabbits. After 13 weeks, histochemical staining suggested that Col I/II blend hydrogels provided favorable conditions for cartilage repair. Histological scoring revealed a statistically higher cartilage repair score for the Col I/II hydrogels compared to either the Col I hydrogels or empty defect controls. Results from this study suggest that there is clinical value in the cartilage repair capabilities of our Col I/II hydrogel with encapsulated MSCs.

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function.

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

Simultaneous intraluminal imaging of tissue autofluorescence and eGFP-labeled cells in engineered vascular grafts inside a bioreactor.

The growing demand for tissue engineered vascular grafts (TEVG) motivates the development of optimized fabrication and monitoring procedures. Bioreactors which provide physiologically-relevant conditions are important for improving holistic TEVG properties and performance. Herein we describe a fiber-based intraluminal imaging system that allows for in situ assessment of vascular materials and re-cellularization processes inside a bioreactor by simultaneous and co-registered measurements of endogenous fluorescence lifetime and exogenous marker fluorescence intensity. The lumen of 6 vascular grafts (∼4 mm diameter) were scanned by reciprocally rotating a 41° angle polished multimode optical fiber inside a protective glass tube with outer diameter of 3 mm. Tubular bovine pericardium constructs were recellularized using enhanced Green Fluorescent Protein (eGFP) transfected cells in a custom bioreactor. The imaging system has resolved consistently the cellular autofluorescence from that of tissue matrix in situ based on the lifetime fluorescence properties of endogenous molecular species. The location of the re-cellularized area was validated by the eGFP emission. Current results demonstrate the potential of this system as a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in cylindrical or other 3-dimensional structures.

Delivery of anti-inflammatory peptides from hollow PEGylated poly(NIPAM) nanoparticles reduces inflammation in an ex vivo osteoarthritis model.

Targeted delivery of anti-inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non-invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N'-bis(acryloyl)cystamine (BAC) cross-linked core out of a non-degradable pNIPAM shell. Sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti-inflammatory mitogen-activated protein kinase-activated protein kinase 2 (MK2)-inhibiting cell-penetrating peptide (KAFAK). The drug-loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro-inflammatory interleukin-6 (IL-6) expression after interleukin-1 beta (IL-1ß) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

An in vitro scaffold-free epithelial-fibroblast coculture model for the larynx.

OBJECTIVES/HYPOTHESIS: Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFß1. STUDY DESIGN: In vitro. METHODS: Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor ß1 (TGFß1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin. RESULTS: Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFß1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture. CONCLUSIONS: These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E185-E192, 2017.

Collagen-binding nanoparticles for extracellular anti-inflammatory peptide delivery decrease platelet activation, promote endothelial migration, and suppress inflammation.

Peripheral artery disease is an atherosclerotic stenosis in the peripheral vasculature that is typically treated via percutaneous transluminal angioplasty. Deployment of the angioplasty balloon damages the endothelial layer, exposing the underlying collagen and allowing for the binding and activation of circulating platelets which initiate an inflammatory cascade leading to eventual restenosis. Here, we report on collagen-binding sulfated poly(N-isopropylacrylamide) nanoparticles that are able to target to the denuded endothelium. Once bound, these nanoparticles present a barrier that reduces cellular and platelet adhesion to the collagenous surface by 67% in whole blood and 59% in platelet-rich plasma under biologically relevant shear rates. In vitro studies indicate that the collagen-binding nanoparticles are able to load and release therapeutic quantities of anti-inflammatory peptides, with the particles reducing inflammation in endothelial and smooth muscle cells by 30% and 40% respectively. Once bound to collagen, the nanoparticles increased endothelial migration while avoiding uptake by smooth muscle cells, indicating that they may promote regeneration of the damaged endothelium while remaining anchored to the collagenous matrix and locally releasing anti-inflammatory peptides into the injured area. Combined, these collagen-binding nanoparticles have the potential to reduce inflammation, and the subsequent restenosis, while simultaneously promoting endothelial regeneration following balloon angioplasty. STATEMENT OF SIGNIFICANCE: In this manuscript, we present our work on the development and characterization of a novel temperature sensitive collagen-binding nanoparticle system. We demonstrate that when bound to a collagenous matrix, the nanoparticles are able to promote endothelial migration while avoiding cellular uptake. We also show that the nanoparticles are able to reduce inflammation via the release of anti-inflammatory peptides which, when combined with its ability to inhibit platelet binding, could lead to reduced intimal hyperplasia following balloon angioplasty. The drug delivery platform presented represents a unique dual therapy biomaterial wherein the nanoparticle itself plays a crucial role in the system's overall therapeutic potential while simultaneously releasing anti-inflammatory peptides.

Controlled release of anti-inflammatory peptides from reducible thermosensitive nanoparticles suppresses cartilage inflammation.

Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.

Synthesis and characterization of a lubricin mimic (mLub) to reduce friction and adhesion on the articular cartilage surface.

The lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; decreased expression of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that is less susceptible to enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a chondroitin sulfate backbone with type II collagen and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated type II collagen and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction, and adhesion, to control levels as assessed over macro-to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction and adhesion, which may suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for treatment in early osteoarthritis following injury.

Prevention of Collagen-Induced Platelet Binding and Activation by Thermosensitive Nanoparticles.

Peripheral artery disease is an atherosclerotic occlusion in the peripheral vasculature that is typically treated via percutaneous transluminal angioplasty. Unfortunately, deployment of the angioplasty balloon damages the endothelial layer, exposing the underlying collagen and allowing for the binding and activation of circulating platelets, which initiate an inflammatory cascade leading to eventual restenosis. Here, we report on the development of poly(NIPAm-MBA-AMPS-AAc) nanoparticles that have a collagen I-binding peptide crosslinked to their surface allowing them to bind to exposed collagen. Once bound, these particles mask the exposed collagen from circulating platelets, effectively reducing collagen-mediated platelet activation. Using collagen I-coated plates, we demonstrate that these particles are able to bind to collagen at concentrations above 0.5 mg/mL. Once bound, these particles inhibit collagen-mediated platelet activation by over 60%. Using light scattering and zeta potential measurements, we investigated the potential of the nanoparticles as a drug delivery platform. We have verified that the collagen-binding nanoparticles retain the temperature sensitivity common to poly(NIPAm)-based nanoparticles while remaining colloidally stable in aqueous environments. We also demonstrate that they are able to passively load and release anti-inflammatory cell penetrating peptides. Combined, we have developed a collagen-binding nanoparticle that has dual therapy potential, preventing collagen-mediated platelet activation while delivering water-soluble therapeutics directly to the damaged area.

Release of anti-inflammatory peptides from thermosensitive nanoparticles with degradable cross-links suppresses pro-inflammatory cytokine production.

Pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) are mediators in the development of many inflammatory diseases. To demonstrate that macrophages take up and respond to thermosensitive nanoparticle drug carriers, we synthesized PEGylated poly(N-isopropylacrylamide-2-acrylamido-2-methyl-1-propanesulfonate) particles cross-linked with degradable disulfide (N,N'-bis(acryloyl)cystamine) (NGPEGSS). An anti-inflammatory peptide (KAFAK) was loaded and released from the thermosensitive nanoparticles and shown to suppress levels of TNF-α and IL-6 production in macrophages. Cellular uptake of fluorescent, thermosensitive, and degradable nanoparticles and therapeutic efficacy of free KAFAK peptide compared to that of KAFAK loaded in PEGylated degradable thermosensitive nanoparticles were examined. The data suggests that the degradable, thermosensitive nanoparticles loaded with KAFAK may be an effective tool to treat inflammatory diseases.

Targeting methicillin-resistant Staphylococcus aureus with short salt-resistant synthetic peptides.

The seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus, and methicillin-resistant Staphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis compared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.

Characterization of endocytic uptake of MK2-inhibitor peptides.

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP-1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP-1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae-mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin-mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP.