Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant To Induce Potent CTLs.

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257-264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (∼6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.

Distribution and Cellular Uptake of PEGylated Polymeric Particles in the Lung Towards Cell-Specific Targeted Delivery.

PURPOSE: We evaluated the role of a poly(ethylene glycol) (PEG) surface coating to increase residence times and alter the cellular fate of nano- and microparticles delivered to the lung. METHODS: Three sizes of PRINT hydrogel particles (80 × 320 nm, 1.5 and 6 µm donuts) with and without a surface PEG coating were instilled in the airways of C57/b6 mice. At time points of 1, 7, and 28 days, BALF and whole lungs were evaluated for the inflammatory cytokine Il-6 and chemokine MIP-2, histopathology, cellular populations of macrophages, dendritic cells (DCs), and granulocytes, and particulate uptake within these cells through flow cytometry, ELISAs, and fluorescent imaging. RESULTS: Particles of all sizes and surface chemistries were readily observed in the lung with minimal inflammatory response at all time points. Surface modification with PEGylation was found to significantly increase lung residence times and homogeneous lung distribution, delaying macrophage clearance of all sizes, with the largest increase in residence time observed for 80 × 320 nm particles. Additionally, it was observed that DCs were recruited to the airway following administration of unPEGylated particles and preferentially associated with these particles. CONCLUSIONS: Pulmonary drug delivery vehicles designed with a PEG surface coating can be used to delay particle uptake and promote cell-specific targeting of therapeutics.

Design of asymmetric particles containing a charged interior and a neutral surface charge: comparative study on in vivo circulation of polyelectrolyte microgels.

Lowering the modulus of hydrogel particles could enable them to bypass in vivo physical barriers that would otherwise filter particles with similar size but higher modulus. Incorporation of electrolyte moieties into the polymer network of hydrogel particles to increase the swelling ratio is a straightforward and quite efficient way to decrease the modulus. In addition, charged groups in hydrogel particles can also help secure cargoes. However, the distribution of charged groups on the surface of a particle can accelerate the clearance of particles. Herein, we developed a method to synthesize highly swollen microgels of precise size with near-neutral surface charge while retaining interior charged groups. A strategy was employed to enable a particle to be highly cross-linked with very small mesh size, and subsequently PEGylated to quench the exterior amines only without affecting the internal amines. Acidic degradation of the cross-linker allows for swelling of the particles to microgels with a desired size and deformability. The microgels fabricated demonstrated extended circulation in vivo compared to their counterparts with a charged surface, and could potentially be utilized in in vivo applications including as oxygen carriers or nucleic acid scavengers.

Using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles.

It has long been hypothesized that elastic modulus governs the biodistribution and circulation times of particles and cells in blood; however, this notion has never been rigorously tested. We synthesized hydrogel microparticles with tunable elasticity in the physiological range, which resemble red blood cells in size and shape, and tested their behavior in vivo. Decreasing the modulus of these particles altered their biodistribution properties, allowing them to bypass several organs, such as the lung, that entrapped their more rigid counterparts, resulting in increasingly longer circulation times well past those of conventional microparticles. An 8-fold decrease in hydrogel modulus correlated to a greater than 30-fold increase in the elimination phase half-life for these particles. These results demonstrate a critical design parameter for hydrogel microparticles.

Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer.

Nanotechnology can provide a critical advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of novel therapeutic delivery vehicles. This paper reports the fabrication of poly(lactic acid-co-glycolic acid)/siRNA nanoparticles coated with lipids for use as prostate cancer therapeutics made via a unique soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT). The PRINT process enables high encapsulation efficiency of siRNA into neutral and monodisperse PLGA particles (32-46% encapsulation efficiency). Lipid-coated PLGA/siRNA PRINT particles were used to deliver therapeutic siRNA in vitro to knockdown genes relevant to prostate cancer.

Low modulus biomimetic microgel particles with high loading of hemoglobin.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.

Potent engineered PLGA nanoparticles by virtue of exceptionally high chemotherapeutic loadings.

Herein we report the fabrication of engineered poly(lactic acid-co-glycolic acid) nanoparticles via the PRINT (particle replication in nonwetting templates) process with high and efficient loadings of docetaxel, up to 40% (w/w) with encapsulation efficiencies >90%. The PRINT process enables independent control of particle properties leading to a higher degree of tailorability than traditional methods. Particles with 40% loading display better in vitro efficacy than particles with lower loadings and the clinical formulation of docetaxel, Taxotere.

Amphiphilic co-networks with moisture-induced surface segregation for high-performance nonfouling coatings.

Herein we report the design of a photocurable amphiphilic co-network consisting of perfluoropolyether and poly(ethylene glycol) segments that display outstanding nonfouling characteristics with respect to spores of green fouling alga Ulva when cured under high humidity conditions. The analysis of contact angle hysteresis revealed that the poly(ethylene glycol) density at the surface was enhanced when cured under high humidity. The nonfouling behavior of nonbiocidal surfaces against marine fouling is rare because such surfaces usually reduce the adhesion of organisms rather than inhibit colonization. We propose that the resultant surface segregation of these materials induced by high humidity may be a promising strategy for achieving nonfouling materials, and such an approach is more important than simply concentrating poly(ethylene glycol) moieties at an interface because the low surface energy has been maintained in our work.

Retinoic Acid-Loaded Poly(lactic-co-glycolic acid) Nanoparticle Formulation of ApoB-100-Derived Peptide 210 Attenuates Atherosclerosis.

We developed a vaccine formulation containing ApoB derived P210 peptides as autoantigens, retinoic acid (RA) as an immune enhancer, both of which were delivered using PLGA nanoparticles. The formula was used to induce an immune response in 12-week-old male Apoe-/- mice with pre-existing atherosclerotic lesions. The nanotechnology platform PRINT® was used to fabricate PLGA nanoparticles that encapsulated RA inside and adsorbed the P210 onto the particle surface. In this study, we demonstrated that immunization of Apoe-/- mice with the formulation was able to considerably attenuate atherosclerotic lesions, accompanied by increased P210 specific IgM and another oxidized lipid derived autoantigen, M2AA, specific IgG autoantibodies, and decreased the inflammatory response, as compared to the P210 group with Freund's adjuvant. Our formulation represents an exciting technology to enhance the efficacy of the P210 vaccine.

Zapped assembly of polymeric (ZAP) nanoparticles for anti-cancer drug delivery.

The starting hypothesis for this work was that microwave synthesis could enable the rapid assembly of polymers into size-specific nanoparticles (NPs). The Zapped Assembly of Polymeric (ZAP) NPs was initially realized using poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) block copolymers and distinct microwave reaction parameters. A library of polymeric NPs was generated with sizes ranging from sub-20 nm to 350 nm and low polydispersity. Select ZAP NPs were synthesized in 30 seconds at different scales and concentrations, up to 200 mg and 100 mg mL-1, without substantial size variation. ZAP NPs with diameters of 25 nm, 50 nm, and 100 nm were loaded with the chemotherapeutic paclitaxel (PXL), demonstrated unique release profiles, and exhibited dose-dependent cytotoxicity similar to Taxol. Incorporation of d-alpha tocopheryl polyethylene glycol succinate (TPGS) and PLGA33k allowed for the production of a sub-40 nm NP with an exceptionally high loading of PXL (12.6 wt%, ca. 7 times the original NP) and a slower release profile. This ZAP NP platform demonstrated scalable, flexible, and tunable synthesis with potential toward clinical scale production of size-specific drug carriers.

Controlling release from 3D printed medical devices using CLIP and drug-loaded liquid resins.

Mass customization along with the ability to generate designs using medical imaging data makes 3D printing an attractive method for the fabrication of patient-tailored drug and medical devices. Herein we describe the application of Continuous Liquid Interface Production (CLIP) as a method to fabricate biocompatible and drug-loaded devices with controlled release properties, using liquid resins containing active pharmaceutical ingredients (API). In this work, we characterize how the release kinetics of a model small molecule, rhodamine B-base (RhB), are affected by device geometry, network crosslink density, and the polymer composition of polycaprolactone- and poly (ethylene glycol)-based networks. To demonstrate the applicability of using API-loaded liquid resins with CLIP, the UV stability was evaluated for a panel of clinically-relevant small molecule drugs. Finally, select formulations were tested for biocompatibility, degradation and encapsulation of docetaxel (DTXL) and dexamethasone-acetate (DexAc). Formulations were shown to be biocompatible over the course of 175 days of in vitro degradation and the clinically-relevant drugs could be encapsulated and released in a controlled fashion. This study reveals the potential of the CLIP manufacturing platform to serve as a method for the fabrication of patient-specific medical and drug-delivery devices for personalized medicine.

Efficacy and pharmacokinetics of a modified acid-labile docetaxel-PRINT(®) nanoparticle formulation against non-small-cell lung cancer brain metastases.

AIM: Particle Replication in Nonwetting Templates (PRINT(®)) PLGA nanoparticles of docetaxel and acid-labile C2-dimethyl-Si-Docetaxel were evaluated with small molecule docetaxel as treatments for non-small-cell lung cancer brain metastases. MATERIALS & METHODS: Pharmacokinetics, survival, tumor growth and mice weight change were efficacy measures against intracranial A549 tumors in nude mice. Treatments were administered by intravenous injection. RESULTS: Intracranial tumor concentrations of PRINT-docetaxel and PRINT-C2-docetaxel were 13- and sevenfold greater, respectively, than SM-docetaxel. C2-docetaxel conversion to docetaxel was threefold higher in intracranial tumor as compared with nontumor tissues. PRINT-C2-docetaxel increased median survival by 35% with less toxicity as compared with other treatments. CONCLUSION: The decreased toxicity of the PRINT-C2-docetaxel improved treatment efficacy against non-small-cell lung cancer brain metastasis.

Biodistribution and Toxicity Studies of PRINT Hydrogel Nanoparticles in Mosquito Larvae and Cells.

Mosquito-borne diseases continue to remain major threats to human and animal health and impediments to socioeconomic development. Increasing mosquito resistance to chemical insecticides is a great public health concern, and new strategies/technologies are necessary to develop the next-generation of vector control tools. We propose to develop a novel method for mosquito control that employs nanoparticles (NPs) as a platform for delivery of mosquitocidal dsRNA molecules to silence mosquito genes and cause vector lethality. Identifying optimal NP chemistry and morphology is imperative for efficient mosquitocide delivery. Toward this end, fluorescently labeled polyethylene glycol NPs of specific sizes, shapes (80 nm x 320 nm, 80 nm x 5000 nm, 200 nm x 200 nm, and 1000 nm x 1000 nm) and charges (negative and positive) were fabricated by Particle Replication in Non-Wetting Templates (PRINT) technology. Biodistribution, persistence, and toxicity of PRINT NPs were evaluated in vitro in mosquito cell culture and in vivo in Anopheles gambiae larvae following parenteral and oral challenge. Following parenteral challenge, the biodistribution of the positively and negatively charged NPs of each size and shape was similar; intense fluorescence was observed in thoracic and abdominal regions of the larval body. Positively charged NPs were more associated with the gastric caeca in the gastrointestinal tract. Negatively charged NPs persisted through metamorphosis and were observed in head, body and ovaries of adults. Following oral challenge, NPs were detected in the larval mid- and hindgut. Positively charged NPs were more efficiently internalized in vitro than negatively charged NPs. Positively charged NPs trafficked to the cytosol, but negatively charged NPs co-localized with lysosomes. Following in vitro and in vivo challenge, none of the NPs tested induced any cytotoxic effects.

Biodistribution and trafficking of hydrogel nanoparticles in adult mosquitoes.

BACKGROUND: Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract. CONCLUSIONS/SIGNIFICANCE: Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.

In vitro and in vivo assessment of targeting lipid-based nanoparticles to the epidermal growth factor-receptor (EGFR) using a novel Heptameric ZEGFR domain.

Lipid-based oil-filled nanoparticles (NPs) with a high concentration of surface-chelated nickel (Ni-NPs) were successfully prepared using a Brij 78-NTA-Ni conjugate synthesized with Brij 78 (Polyoxyethylene (20) stearyl ether) and nitrilotriacetic acid (NTA). The facile incorporation of the Brij 78-NTA-Ni conjugate into the NP formulation allowed up to 90% Ni incorporation, which was a significant improvement over the previously used standard agent DOGS-NTA-Ni which led to ~6% Ni incorporation. The Ni-NPs were targeted to the highly epidermal growth factor receptor (EGFR)-overexpressing epidermoid carcinoma cells A431. This was accomplished using a novel high affinity histidine×6-tagged EGFR-binding Z domain (heptameric Z(EGFR) domain). In vitro cell uptake studies showed enhanced internalization (up to 90%) of the targeted Ni-NPs in A431 cells with only ≤10% internalization of the untargeted Ni-NPs. ICP-MS analysis used to quantify the amount of Ni in the cells were in close agreement with flow cytometry studies, which showed a dose dependent increase in the amount of Ni with the targeted Ni-NPs. Cell uptake competition studies showed that internalization of the targeted Ni-NPs within the cells was competed off with free heptameric Z(EGFR) domain at concentrations of 8.75ng/mL or higher. In vivo studies were carried out in nude mice bearing A431 tumors to determine the biodistribution and intracellular delivery. Near Infrared (NIR) optical imaging studies using Alexa750-labeled heptameric Z(EGFR) domain showed localization of 19% of the total detected fluorescence intensity in the tumor tissue, 28% in the liver and 42% in the kidneys 16h post i.v. injection. ICP-MS analysis showed almost a two-fold increase in the amount of intracellular Ni with the targeted Ni-NPs. These new Ni-NPs could be a very useful tool for targeting and drug delivery to a wide range of EGFR positive cancers.