Background: Glucocorticoids are an important class of therapeutics used in a variety of applications, including allotransplantations. Dexamethasone (Dexa) is well-known for its strong anti-inflammatory, immunosuppressive, and anticancer properties. However, its clinical use is often limited by its poor water solubility, poor pharmacokinetics, and high likelihood of systemic side effects. Methods: To address the issues, we tested a combined strategy where our original Drug-Integrating Amphiphilic Nano-Assemblies (DIANAs), a class of self-assembling polymeric nanoparticles designed for controlled drug release, were used to solubilize and deliver dexamethasone palmitate (DexP), a hydrophobic prodrug of dexamethasone. Results: The palmitate chains of the prodrug can form strong van der Waals interactions with the hydrophobic moieties of the PEG-PPS block copolymer used here. In water, this resulted in the self-assembling of stable dexamethasone palmitate–PEG–PPS nanomicelles, termed DexP-nMICs, with a 25 nm average diameter that slowly released Dexa over more than two weeks. Conclusions: Here we demonstrated that DexP-nMICs can carry elevated amounts of Dexa—increasing its solubility in water—prolong circulation in its pharmacologically active form in vivo and provide passive targeting to inflammation sites. The anti-inflammatory efficacy of DexP-nMICs was first confirmed in vitro on stimulated macrophages, demonstrating a significant reduction in cytokine secretion. An allogeneic mouse skin transplant model, used to assess the therapeutic potential of DexP-nMICs in vivo, confirmed its ability to provide graft-targeted delivery and prolong graft survival as compared to the unformulated parent drug. Therefore, DexP-nMICs are a promising candidate for sustained and localized use of anti-inflammatory drugs in cell and tissue transplantations.

The need for chronic systemic immunosuppression, which is associated with unavoidable side-effects, greatly limits the applicability of allogeneic cell transplantation for regenerative medicine applications including pancreatic islet cell transplantation to restore insulin production in type 1 diabetes (T1D). Cell transplantation in confined sites enables the localized delivery of anti-inflammatory and immunomodulatory drugs to prevent graft loss by innate and adaptive immunity, providing an opportunity to achieve local effects while minimizing unwanted systemic side effects. Nanoparticles can provide the means to achieve the needed localized and sustained drug delivery either by graft targeting or co-implantation. Here, we evaluated the potential of our versatile platform of drug-integrating amphiphilic nanomaterial assemblies (DIANAs) for targeted drug delivery to an inflamed site model relevant for islet transplantation. We tested either passive targeting of intravenous administered spherical nanomicelles (nMIC; 20–25 nm diameter) or co-implantation of elongated nanofibrils (nFIB; 5 nm diameter and >1 μm length). To assess the ability of nMIC and nFIB to target an inflamed graft site, we used a lipophilic fluorescent cargo (DiD and DiR) and evaluated the in vivo biodistribution and cellular uptake in the graft site and other organs, including draining and non-draining lymph nodes, after systemic administration (nMIC) and/or graft co-transplantation (nFIB) in mice. Localized inflammation was generated either by using an LPS injection or by using biomaterial-coated islet-like bead implantation in the subcutaneous site. A cell transplant inflammation model was used as well to test nMIC- and nFIB-targeted biodistribution. We found that nMIC can reach the inflamed site after systemic administration, while nFIB remains localized for several days after co-implantation. We confirmed that DIANAs are taken up by different immune cell populations responsible for graft inflammation. Therefore, DIANA is a useful approach for targeted and/or localized delivery of immunomodulatory drugs to decrease innate and adaptive immune responses that cause graft loss after transplantation of therapeutic cells.

Type-I Diabetes (T1D) is caused by defective immunotolerance mechanisms enabling autoreactive T cells to escape regulation in lymphoid organs and destroy insulin-producing β-cells in the pancreas, leading to insulin dependence. Strategies to promote β-cell tolerance could arrest T1D. We previously showed that secretion of secondary lymphoid chemokine CCL21 by CCL21 transgenic β-cells induced tolerance and protected non-obese diabetic (NOD) mice from T1D. T1D protection was associated with formation of lymph node-like stromal networks containing tolerogenic fibroblastic reticular cells (FRCs). Here, we developed a polyethylene glycol (PEG) hydrogel platform with hydrolytically degradable PEG-diester dithiol crosslinkers to provide controlled and sustained delivery of CCL21 and β-cell antigens for at least 28 days in vitro and recapitulate properties associated with the tolerogenic environment of CCL21 transgenic β-cells in our previous studies. CCL21 and MHC-II restricted antigens were tethered to gels via simple click-chemistry while MHC-I restricted antigens were loaded in PEG-based polymeric nanovesicles and incorporated in the gel networks. CCL21 and antigen release kinetics depended on the PEG gel tethering strategy and the linkers. Importantly, in vitro functionality, chemotaxis, and activation of antigen-specific T cells were preserved. Implantation of CCL21 and β-cell antigen gels under the kidney capsule of pre-diabetic NOD mice led to enrichment of adoptively transferred antigen-specific T cells, formation of gp38 + FRC-like stromal cell networks, and increased regulation of specific T cells with reduced accumulation within pancreatic islets. Thus, our platform for sustained release of β-cell antigens and CCL21 immunomodulatory molecule could enable the development of antigen-specific tolerance therapies for T1D.

Islet transplantation has been established as a viable treatment modality for type 1 diabetes. However, the side effects of the systemic immunosuppression required for patients often outweigh its benefits. Here, we engineer programmed death ligand-1 and cytotoxic T lymphocyte antigen 4 immunoglobulin fusion protein–modified mesenchymal stromal cells (MSCs) as accessory cells for islet cotransplantation. The engineered MSCs (eMSCs) improved the outcome of both syngeneic and allogeneic islet transplantation in diabetic mice and resulted in allograft survival for up to 100 days without any systemic immunosuppression. Immunophenotyping revealed reduced infiltration of CD4 + or CD8 + T effector cells and increased infiltration of T regulatory cells within the allografts cotransplanted with eMSCs compared to controls. The results suggest that the eMSCs can induce local immunomodulation and may be applicable in clinical islet transplantation to reduce or minimize the need of systemic immunosuppression and ameliorate its negative impact.

Immunomodulatory therapies are limited by unavoidable side effects as well as poor solubility, stability, and pharmacokinetic properties. Nanomaterial-based drug delivery may overcome these limitations by increasing drug solubility, site-targeting, and duration of action. Here, we prepared innovative drug-integrating amphiphilic nanomaterial assemblies (DIANA) with tunable hydrophobicity, size, and morphology, and we evaluated their ability to deliver cyclosporine A (CsA) for immunomodulatory applications. We synthesized amphiphilic block copolymers made of poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS) and poly(ethylene glycol)-oligo(ethylene sulfide) (PEG-OES) that can self-assemble into solid core nanomicelles (nMIC, with ≈20 nm diameter) and nanofibrils (nFIB, with ≈5 nm diameter and > 500 nm length), respectively. nMIC and nFIB displayed good CsA encapsulation efficiency (up to 4.5 and 2 mg/mL, respectively in aqueous solution), superior to many other solubilization methods, and provided sustained release (>14 and > 7 days for the nMIC and nFIB) without compromising CsA's pharmacological activity. Treatment of insulin-secreting cells with unloaded DIANAs did not impair cell viability and functionality. Both CsA-loaded DIANAs inhibited the proliferation and activation of insulin-reactive cytotoxic T cells in vitro. Subcutaneous injections of CsA-loaded DIANAs in mice provided CsA sustained release, decreasing alloantigen-induced immune responses in the draining lymph node at lower doses and reduced administration frequency than unformulated CsA. While nMIC solubilized higher amounts and provided more sustained release of CsA in vitro, nFIB enhanced cellular uptake and promoted local retention due to slower trafficking in vivo. DIANAs provide a versatile platform for a local immune suppression regimen that can be applied to allogeneic cell transplantation.

In this work, we report the synthesis of novel fluorescent molecules, based on 1,8-naphthalimide thio- and amino-derivatives, designed to display an OFF-ON and ON-OFF photoelectron transfer fluorescence switch upon interaction with a proton-donor drug. We have functionalized the new probes to allow easy formation of a covalent link to polymer matrices, for future applications as drug delivery sensors. We have investigated the fluorescent switch of the new probes using flufenamic acid (FA, pKa 3.65), a water insoluble, non-steroidal anti-inflammatory drug, as a model drug and proton source. The rapid interaction of the new probes with FA resulted in fluorescence enhancement or decrease and a large Stokes shift, all of which allowed the detection of the drug in the nanomolar range. The new 1,8-naphthalimide fluorescent dyes reported in this work are interesting tools for the detection and quantification of acidic drugs and biomolecules.

Mycolic acid (MA), a major lipid component of Mycobacterium tuberculosis (Mtb) cell wall, can be presented by the non-polymorphic antigen presenting molecule CD1b to T cells isolated from Mtb-infected individuals. These MA-specific CD1b-restricted T cells are cytotoxic, produce Th1 cytokines, and form memory populations, suggesting that MA can be explored as a potential subunit vaccine candidate for TB. However, the controlled elicitation of MA-specific T cell responses has been challenging due to difficulties in the targeted delivery of lipid antigens and a lack of suitable animal models. In this study, we generated MA-loaded micellar nanocarriers (MA-Mc) comprised of self-assembled poly(ethylene glycol)-bl-poly(propylene sulfide; PEG-PPS) copolymers conjugated to an acid sensitive fluorophore to enhance intracellular delivery of MA to phagocytic immune cells. Using humanized CD1 transgenic (hCD1Tg) mice, we found these nanobiomaterials to be endocytosed by bone marrow-derived dendritic cells (DCs) and localized to lysosomal compartments. Additionally, MA-Mc demonstrated superior efficacy over free MA in activating MA-specific TCR transgenic (DN1) T cells in vitro. Following intranasal immunization, MA-Mc were primarily taken up by alveolar macrophages and DCs in the lung and induced activation and proliferation of adoptively transferred DN1 T cells. Furthermore, intranasal immunization with MA-Mc induced MA-specific T cell responses in the lungs of hCD1Tg mice. Collectively, our data demonstrates that pulmonary delivery of MA via PEG-PPS micelles to DCs can elicit potent CD1b-restricted T cell responses both in vitro and in vivo and MA-Mc could be explored as subunit vaccines against Mtb infection.

Immune responses play a key role in maintaining tissue homeostasis, influencing nearly all organs and systems of the body including skin, gut, lungs, brain, and the cardiovascular system.Excessive or dysregulated immune responses and chronic inflammation represent a central driving force in many disorders, including infectious, inflammatory, and autoimmune diseases, as well as cancer.Cells of the mononuclear phagocyte lineage act as innate sentinels and are actively involved in regulating the balance between homeostasis and inflammation, thus ultimately contributing to the maintenance of the health condition.Age-and gender-related differences in immune response as well as in gut microbiota are emerging as important contributors in increasing the complexity in the diagnosis, treatment, and prevention of immune-mediated disorders.In four original investigation articles, different autoimmune diseases were studied to find new biomarkers that could help explain the aetiology and pathogenesis of the diseases and be useful for new targeted therapy.Myasthenia gravis (MG) is an antibody-mediated disease affecting the neuromuscular junction, caused by antibodies against the nicotinic acetylcholine receptor (AChR, AChR-Ab).Thanks to short half-life serum levels, free immunoglobulin light chains (FLCs) can be considered an instantaneous marker of B cell activity.In their study, U. Basile et al. showed an increase in free k chains in both AChR-and muscle-specific tyrosine kinase (MuSK-) MG while free λ chain levels were increased only in AChR-MG.Thus, they suggest that at least k chains can be considered a very sensitive circulating biomarker of B cell

Drug delivery emerged as a discipline to solve problems associated with the majority of the current drugs, such as poor water solubility, poor physical stability, poor absorption and side effects. Large pharmaceutical companies are investing in drug delivery technologies to find better ways to administer existing drugs rather than designing new products. The secret to a successful drug delivery system, one that allows controlled release over a prolonged period of time, is in the carrier. An ideal drug carrier must be biocompatible, biodegradable, water friendly, selective, easy to prepare, stable, cheap, and finally, ultra-small. Therefore, the interdisciplinary field of nanotechnology and nanomaterials is playing a big role in drug delivery by providing new tools to develop ideal nanocarriers and find appropriate solutions for medical problems. In this review, we briefly recapitulate the history of nanomaterials in drug delivery, explore their unique properties, and report an example of the design and development of polymer-based nanomaterials. We also revisit the most challenging applications of drug delivery for cancer treatment, cell and tissue transplantations and stem cell therapies. Overall, nanotechnology-based drug delivery systems administered by different routes can be considered promising tools to improve patient compliance and achieve better therapeutic outcomes in critical illnesses.

Event Abstract Back to Event Development and evaluation of self-assembled biomaterials to maximise the function of conformal encapsulation of pancreatic islets in type 1 diabetes Diana Velluto1, Vita Manzoli1, 2 and Alice A. Tomei1, 3 1 Miller School of Medicine, University of Miami, Diabetes Research Institute, United States 2 Politecnico di Milano, Electronics, Information and Bioengineering, Italy 3 University of Miami, Biomedical Engineering, United States T1D is an autoimmune disease that affects over three million Americans. In T1D, beta cells within pancreatic islets are selectively destroyed by autoimmune responses. Beta cells are responsible for secreting insulin, which regulates glucose metabolism and homeostasis. Patients with T1D become dependent on exogenous insulin injections and are susceptible to acute and chronic complications, which can be life-threatening. Islet transplantation completely eliminates complications of type-1 diabetes (T1D) and can restore insulin secretion and glucose homeostasis, but requires life-long immunosuppression and graft survival is limited. Islet encapsulation may allow transplantation without immunosuppression but it failed in the past three decades. Here we combine a recently developed encapsulation techniques (conformal coating, CC)[1] and new nanotechnology strategies to create an encapsulation platform for islets transplantation without immunosuppression and with enhanced cells survival. In particular I have previously developed PEG-PPS (polyethylene glycol-polypropylene sulfide) amphiphilic block copolymers (Fig. 1) that carry and deliver hydrophobic drugs, in vitro and in vivo, including dexamethasone (DEXA) and cyclosporine-A (CsA)[2],[3]. DEXA and CsA are approved by the FDA for utilization in islet transplantation and their synergistic effect with islet encapsulation has been proven. Additionally, I showed that PEG-PPS block copolymers are non-immunogenic and non-toxic. To increase the stability of amphiphilic block copolymers upon oxidation, I recently developed and patented a new family of block copolymers made of PEG-OES (polyethylene glycol-oligoethylene sulfide)[4]. The combination of hydrophobicity and crystallinity of PEG-OES blocks drives self-assembling into highly stable linear-fibrils (nano-fibers, Fig. 2A). I found that also PEG-OES nano-fibers allow rapid and stable incorporation of CsA and DEXA without the need for chemical conjugation. The nanofibers and their cargo can be incorporated within CC capsules of the islet before transplantation (Fig. 2B) without compromising GSIR functionality of enclosed human islets. Like PEG-PPS micelles, PEG-OES nano-fibers are able of slowly releasing CsA and DEXA by diffusion, oxidation or reduction in vivo, providing sustained local immunomodulation in the encapsulated graft. PEG-OES nano-fibers are also used here as carriers of perfluorocarbons (PFCs) to increase oxygen diffusivity in the encapsulated islets (Fig. 2). PFCs can dissolve significant quantities of O2 with higher affinity than hemoglobin. PFCs are hydrophobic and therefore they can be incorporated into the hydrophobic core of PEG-OES nano-fibers, promoting the transport of O2 into the CC islets and avoiding any systemic toxicity. The work presented here is intended to improve the existing CC technology to allow its translation into human clinical trials, balancing nutrient transport and immunoisolation of CC capsules through PEG-OES nanocarriers. References:[1] Tomei AA, Manzoli V, Fraker CA, Giraldo J, Velluto D, Najjar M, et al. Device design and materials optimization of conformal coating for islets of Langerhans. Proc Natl Acad Sci U S A 2014 ;111(29):10514-9.[2] Velluto D, Demurtas D, Hubbell JA. PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example. Molecular pharmaceutics 2008; 5(4):632-42[3] Dane KY, Nembrini C, Tomei AA, Eby JK, O'Neil CP, Velluto D, et al. Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival. Journal of controlled release : official journal of the Controlled Release Society 2011; 156(2):154-60.[4] Brubaker CE, Velluto D, Demurtas D, Phelps EA, Hubbell JA. Crystalline Oligo(ethylene sulfide) Domains Define Highly Stable Supramolecular Block Copolymer Assemblies. ACS nano 2015; 9(7):6872-81. Keywords: Drug delivery, nanofiber, nanoparticle, Polymeric material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials for therapeutic delivery Citation: Velluto D, Manzoli V and Tomei AA (2016). Development and evaluation of self-assembled biomaterials to maximise the function of conformal encapsulation of pancreatic islets in type 1 diabetes. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02636 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Diana Velluto Vita Manzoli Alice A Tomei Google Diana Velluto Vita Manzoli Alice A Tomei Google Scholar Diana Velluto Vita Manzoli Alice A Tomei PubMed Diana Velluto Vita Manzoli Alice A Tomei Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

In this paper, we report on a potential cancer drug delivery system that utilizes the ligand targeting of the folate receptor. Our drug delivery system consists of Pluronic-P105 micelles, targeted with folic acid moieties. A melanoma folate positive (FR+) (B16-F10), and a fibroblast folate negative (FR-) (NIH-3T3) cell lines are used to compare the cellular accumulation of a chemotherapeutic drug (Doxorubicin) when the delivery is mediated by folated Pluronic P105 micelles. In order to obtain a proper comparison, we corrected for the quenching of Doxorubicin by folic acid molecules and illustrated the significant effect of quenching on the analysis of similar systems. Results show an 80% increase in the accumulation of the antineoplastic agent in the FR+ cell line, when compared to the FR- cell line, thus providing evidence that the efficacy of Pluronic micelles, as drug delivery vehicles, can be enhanced via folic acid targeting.

With proper control over copolymer design and solvation conditions, self-assembled materials display impressive morphological variety that encompasses nanoscale colloids as well as bulk three-dimensional architectures. Here we take advantage of both hydrophobicity and crystallinity to mediate supramolecular self-assembly of spherical micellar, linear fibrillar, or hydrogel structures by a family of highly asymmetric poly(ethylene glycol)-b-oligo(ethylene sulfide) (PEG-OES) copolymers. Assembly structural polymorphism was achieved with modification of PEG-OES topology (linear versus multiarm) and with precise, monomer-by-monomer control of OES length. Notably, all three morphologies were accessed utilizing OES oligomers with degrees of polymerization as short as three. These exceptionally small assembly forming blocks represent the first application of ethylene sulfide oligomers in supramolecular materials. While the assemblies demonstrated robust aqueous stability over time, oxidation by hydrogen peroxide progressively converted ethylene sulfide residues to increasingly hydrophilic and amorphous sulfoxides and sulfones, causing morphological changes and permanent disassembly. We utilized complementary microscopic and spectroscopic techniques to confirm this chemical stimulus-responsive behavior in self-assembled PEG-OES colloidal dispersions and physical gels. In addition to inherent stimulus-responsive behavior, fibrillar assemblies demonstrated biologically relevant molecular delivery, as confirmed by the dose-dependent activation of murine bone marrow-derived dendritic cells following fibril-mediated delivery of the immunological adjuvant monophosphoryl lipid A. In physical gels composed of either linear or multiarm PEG-OES precursors, rheologic analysis also identified mechanical stimulus-responsive shear thinning behavior. Thanks to the facile preparation, user-defined morphology, aqueous stability, carrier functionality, and stimuli-responsive behaviors of PEG-OES supramolecular assemblies, our findings support a future role for these materials as injectable or implantable biomaterials.

Intracellular protein (BSA) delivery by a phosphonate@mesoporous silica nanoparticle vehicle, PMSN, with high load capacity for the relatively large test protein BSA, is described. Wide pore (11.6 nm) PMSN nanoparticles were synthesised and loaded with a BSA cargo to give BSA#@PMSN*, where # and * signify Fluorescein and Rhodamine fluorescent labels respectively. Internalisation of BSA#@PMSN*s by HeLa cells was analysed from confocal microscopy and TEM images after dose and time dependent treatments. No evidence of cytotoxicity was observed after 24 h and in contrast to PMSN* no significant loss of BSA#@PMSN* was observed after 3 h incubation of the loaded cells in DMEM. Receptor blocking experiments showed caveolar uptake of PMSN* and folate receptor mediated uptake of BSA#@PMSN*s.

To minimize the adverse side effects of conventional chemotherapy, a targeted micellar drug carrier was investigated that retains hydrophobic drugs in its core and then releases the drug via ultrasonic activation. This paper compares the percent drug release from folated versus non-folated micelles by insonation at 70 kHz and different acoustic power densities. The encapsulated drug is Doxoru- bicin (Dox). A physical model of zero-order release with first-order re-encapsulation was used to fit the experimental kinetic data. Additionally, the acoustic activation power density and Gibbs free energy were introduced and calculated for folated and non-targeted micelles. The data suggests an important role of inertial cavitation in drug release and the presence of a power density threshold for inertial cavitation.

Significance Cell encapsulation with biocompatible and permeable hydrogels may allow transplantation without immunosuppression. As an alternative to standard microencapsulation approaches that create single-sized capsules around cell clusters of different sizes, we have designed and optimized a novel approach for conformal coating of islets of Langerhans, resulting in thin, complete, and uniform coatings of similar thickness on differently sized islets. Coated islets exhibited no delay in glucose-stimulated insulin release or loss of function during culture, which is often observed with naked islets. The conformal coating reduces transplant volume relative to traditional encapsulation approaches. When transplanted in syngeneic diabetic mice, conformally coated islets restored and maintained euglycemia for more than 100 d with no foreign body reaction and normal revascularization.

This paper reports the synthesis of large pore (11 nm) monodisperse raspberry textured phosphonate@silica nanoparticles (70-90 nm) with high capacity for protein immobilization. The raspberry nanoparticles denoted RNP_PME(2.5) with phosphonate loading 2.5 mmol g(-1), formed using an organosilanephosphonate (MeO)(3)SiCH2CH2PO(OMe)(2), as silica surface modifier and structure directing agent. Specific reaction conditions including temperature and concentration of phosphonate, base, surfactant and co-solvent were required for RNP_PME(2.5) formation. Rhodamine B labelled RNP_PME(2.5) was readily internalised by HeLa cells with no deficit of cell viability. Aqueous dispersions of RNP_PME(2.5) were stable over several months. In protein immobilization studies using BSA, bovine serum albumin, with RNP_PME(2.5), smaller pore (similar to 3 nm) phosphonate@silica nanoparticles NP_PME(1.0) and NP_PME(0.2) and mesoporous silica nanoparticles, MSN, the large pore RNP_PME(2.5) gave highest BSA loading 266 mg g(-1), formed the most stable aqueous dispersions (BSA@MSN was unstable and precipitated) and gave the best protection against BSA structural distortion at pH 7.4.