Drug Integrating Amphiphilic Nano-Assemblies: 2. Spatiotemporal Distribution within Inflammation Sites.

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.

Engineered immunomodulatory accessory cells improve experimental allogeneic islet transplantation without immunosuppression.

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.

CCL21 and beta-cell antigen releasing hydrogels as tolerance-inducing therapy in Type I diabetes.

Type-I Diabetes (T1D) is caused by defective immunotolerance mechanisms enabling autoreactive T cells to escape regulation in lymphoid organs and destroy insulin-producing ß-cells in the pancreas, leading to insulin dependence. Strategies to promote ß-cell tolerance could arrest T1D. We previously showed that secretion of secondary lymphoid chemokine CCL21 by CCL21 transgenic ß-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 ß-cell antigens for at least 28 days in vitro and recapitulate properties associated with the tolerogenic environment of CCL21 transgenic ß-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 ß-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 ß-cell antigens and CCL21 immunomodulatory molecule could enable the development of antigen-specific tolerance therapies for T1D.

Drug-Integrating Amphiphilic Nanomaterial Assemblies: 1. Spatiotemporal control of cyclosporine delivery and activity using nanomicelles and nanofibrils.

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.

Induction of Mycobacterium Tuberculosis Lipid-Specific T Cell Responses by Pulmonary Delivery of Mycolic Acid-Loaded Polymeric Micellar Nanocarriers.

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.

Targeting the Folate Receptor: Effects of Conjugating Folic Acid to DOX Loaded Polymeric Micelles.

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.

Kinetics of Ultrasonic Drug Delivery from Targeted Micelles.

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.

Crystalline Oligo(ethylene sulfide) Domains Define Highly Stable Supramolecular Block Copolymer Assemblies.

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 delivery of BSA by phosphonate@silica nanoparticles.

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.

Device design and materials optimization of conformal coating for islets of Langerhans.

Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600-1,000 µm in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture.

Large pore raspberry textured phosphonate@silica nanoparticles for protein immobilization.

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)3SiCH2CH2PO(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 (∼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.

Hierarchical self-assembly of amphiphilic calix[6]arene wheels and viologen axles in water.

We have designed and synthesized two amphiphilic calix[6]arene derivatives, CA8 and CA18, that combine the potential to act as wheel components for pseudorotaxane structures with the self-assembly features typical of surfactant molecules in aqueous solution. Their endo-cavity recognition and selfaggregation properties were compared with those of a non-amphiphilic analogue, C8. TEM, DLS, and fluorescence experiments show that in water the amphiphilic calixarenes form vesicle- and micelle-like aggregates. The size, nature and properties of such aggregates depend on the length of the alkyl chain anchored at the lower rim of the calix[6]arene skeleton, as well as on the inclusion of a molecular guest into the wheel. Specifically, the release of a fluorescent guest entrapped inside the CA8 vesicles is accelerated in the presence of dioctylviologen axles that can pierce the calixarene cavity.

Investigating the acoustic release of doxorubicin from targeted micelles.

The main problem associated with the administration of anti-cancer medication is that the drug is delivered throughout the body causing undesirable side effects. Therefore, it is important to synthesize drug carriers capable of minimizing the adverse side effects of chemotherapy by preferentially targeting tumor cells both actively (e.g. a folate receptor) and using external stimulus (e.g. ultrasound). In this paper, we report the synthesis of Pluronic P105 micelles with a folate targeting moiety (with a yield of 48%) containing doxorubicin (Dox). We applied low frequency ultrasound as an external stimulus and measured the amount of release of Dox from these folated micelles. The results showed that the percent drug release increases as the power intensity of ultrasound increases. The maximum amount of release (14%) was measured at 5.4 W/cm(2). A power density threshold at approximately 0.55 W/cm(2) exists below which no statistically significant release was observed. This lower threshold suggests that cavitation plays an important role in triggering drug release from targeted micelles.

Sorting live stem cells based on Sox2 mRNA expression.

While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

PEG-b-PPS-b-PEI micelles and PEG-b-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors for plasmid DNA: tumor immunotoxicity in B16F10 melanoma.

Cationic micelles formed from poly(ethylene glycol)-bl-poly(propylene sulfide)-bl-poly(ethylene imine) (PEG-b-PPS-b-PEI) and from mixtures of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-b-PPS) with PEG-b-PPS-b-PEI were explored as non-viral vectors for plasmid DNA (pDNA) transfection in a tumor immunotoxicity model. Complexes with pDNA were found to be templated exclusively by the size of the pDNA-free micelles and ranged from 240 nm (for PEG-b-PPS-b-PEI) to 30 nm (for mixed micelles of PEG-b-PPS/PEG-b-PPS-b-PEI). Both formulations transfected melanoma cells well in vitro. As a model with a functional read-out of tumor cell death, one with likely only small bystander effects, tumors were transfected with an antigen transgene, using an antigen to which the recipient animals had been previously vaccinated with a Th1-biasing adjuvant. Reduction in tumor growth, increase in intratumoral infiltration of cytotoxic T lymphocytes and accumulation of Th1-biasing cytokines indicated that both micelle formulations transfected efficiently compared with naked pDNA and with low cytotoxicity.

Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival.

By delivering immunomodulatory drugs in vivo directly to lymph nodes draining an injection site, an opportunity exists to increase drug bioavailability to local immune cells. Importantly, particles smaller than 100 nm are efficiently transported through lymphatic vessels to draining lymph nodes. To investigate whether this approach could be used for local delivery of immunomodulatory drugs, amphiphilic poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) block copolymers forming 50 nm micelles were used to encapsulate hydrophobic drugs. Micelle drainage was determined using fluorescent micelles and showed effective targeting of multiple immune cell subsets in lymph nodes. For functional studies of our formulations, two approaches were considered. To evaluate the efficacy of anti-inflammatory drug delivery, dendritic cell activation was shown to be prevented when mice were pretreated with micelles loaded with the glucocorticoid mometasone and then challenged with the TLR9 ligand, CpG. To evaluate whether immunosuppressive drug-loaded micelles were effective in prolonging MHC-mismatched allograft survival, BALB/c mice were treated for 14 consecutive days with drug-loaded micelles following transplantation of allogenic C57BL/6 tail skin. Micelles loaded with a mixture of rapamycin and tacrolimus prolonged allograft survival by 2-fold. Our results indicate that the drug-loaded micelle approach effectively targets the draining lymph nodes and exhibits proper immune regulation.

Characterization of cationic liposomes. Influence of the bilayer composition on the kinetics of the liposome breakdown.

The cationic large unilamellar mixed liposomes from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and didodecyldimethylammonium bromide (DDAB) or dioctadecyldimethylammonium bromide (DODAB) were prepared. The influence of the addition of Triton X-100 (TX-100) or octaethylene glycol mono-n-dodecylether (C(12)E(8)) on the membrane integrity was investigated turbidimetrically. The stability of the liposomal systems was estimated by monitoring fluorimetrically at 25 °C the rate of spontaneous and surfactant-induced release of entrapped 5(6)-carboxyfluorescein (CF). In order to evaluate the interaction of the cationic DODAB guest with the host POPC membrane, the main phase transition temperatures (T(m)) were determined by electron paramagnetic resonance spectroscopy (EPR). All the results obtained show that the presence of DODAB and DDAB stabilizes the POPC liposomes. The extent of stabilization depends on the concentration and nature of the cationic guest.

Use of simple kinetic and reaction-order measurements for the evaluation of the mechanism of surfactant-liposome interactions.

Surfactant-liposome interactions have been previously studied through different methods and techniques. We present here a classical physical chemistry study on liposome solutions added to destabilizing agents at concentrations well above the solubilization concentration, which enable us to draw useful and interesting conclusions about the mechanism of surfactant-induced liposomal breakdown by simply exploiting the kinetics and the reaction order of the liposomal content release. In such excess of surfactant, the mechanism of surfactant-induced rupture of the liposomes has been demonstrated to be different from that proposed for low surfactant concentrations. Thus, depending on the surfactant concentration, two prevailing processes have been evidenced: (i) a cooperative mechanism that implies the assembly of a critical number of surfactant molecules to trigger the formation of a channel and therefore the release of the liposomal content and (ii) a mechanism driven by direct interaction of the surfactant molecules with the lipids that causes the complete solubilization of the liposomes. The former mechanism occurs at low surfactant concentrations, whereas the latter occurs at higher concentrations and above the CMC of the surfactants. The effect of different guests embedded into the liposomal bilayer on the mechanism of surfactant-induced liposomal breakdown has been compared by using the second-order rate constants measured for the liposome breakdown process.

Biocompatible dispersions of carbon nanotubes: a potential tool for intracellular transport of anticancer drugs.

The use of the biocompatible amphiphilic diblock copolymer poly(ethylene glycol-b-propylene sulfide) (PEG44PPS20) allows a tuned loading of doxorubicin onto the surface of non-functionalized multi-walled carbon nanotubes and an efficient cell internalization. The obtained multi-walled carbon nanotube-based systems show enhanced cytotoxic activity with respect to non-vehicled doxorubicin.