Iontophoretic device delivery for the localized treatment of pancreatic ductal adenocarcinoma.

Poor delivery and systemic toxicity of many cytotoxic agents, such as the recent promising combination chemotherapy regimen of folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), restrict their full utility in the treatment of pancreatic cancer. Local delivery of chemotherapies has become possible using iontophoretic devices that are implanted directly onto pancreatic tumors. We have fabricated implantable iontophoretic devices and tested the local iontophoretic delivery of FOLFIRINOX for the treatment of pancreatic cancer in an orthotopic patient-derived xenograft model. Iontophoretic delivery of FOLFIRINOX was found to increase tumor exposure by almost an order of magnitude compared with i.v. delivery with substantially lower plasma concentrations. Mice treated for 7 wk with device FOLFIRINOX experienced significantly greater tumor growth inhibition compared with i.v. FOLFIRINOX. A marker of cell proliferation, Ki-67, was stained, showing a significant reduction in tumor cell proliferation. These data capitalize on the unique ability of an implantable iontophoretic device to deliver much higher concentrations of drug to the tumor compared with i.v. delivery. Local iontophoretic delivery of cytotoxic agents should be considered for the treatment of patients with unresectable nonmetastatic disease and for patients with the need for palliation of local symptoms, and may be considered as a neoadjuvant approach to improve resection rates and outcome in patients with localized and locally advanced pancreatic cancer.

Optimization of Surface Display of DENV2 E Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses.

The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.

Synthesis and Characterization of Cetuximab-Docetaxel and Panitumumab-Docetaxel Antibody-Drug Conjugates for EGFR-Overexpressing Cancer Therapy.

The safety and efficacy of anticancer antibody-drug conjugates (ADCs) depend on the selection of tumor-targeting monoclonal antibody (mAb), linker, and drug, as well as their specific chemical arrangement and linkage chemistry. In this study, we used a heterobifunctional cross-linker to conjugate docetaxel (DX) to cetuximab (CET) or panitumumab (PAN). The resulting ADCs were investigated for their in vitro EGFR-specific cytotoxicity and in vivo anticancer activity. Reaction conditions, such as reducing agent, time, temperature, and alkylation buffer, were optimized to yield potent and stable ADCs with consistent batch-to-batch drug-to-antibody ratios (DARs). ADCs were synthesized with DARs from 0.4 to 3.0, and all retained their EGFR affinity and specificity after modification. ADCs were sensitive to cell surface wildtype EGFR expression, demonstrating more cytotoxicity in EGFR-expressing A431 and MDA-MB-231 cell lines compared to U87MG cells. A431 tumor-bearing mice treated once weekly for four weeks with 100 mg/kg cetuximab-docetaxel ADC (C-SC-DX, DAR 2.5) showed durable anticancer responses and improved overall survival compared to the same treatment regimen with 1 mg/kg DX, 100 mg/kg CET, or a combination 1 mg/kg DX and 100 mg/kg CET. New treatment options are emerging for patients with both wild-type and mutated EGFR-overexpressing cancers, and these studies highlight the potential role of EGFR-targeted ADC therapies as a promising new treatment option.

Formulation of High-Performance Dry Powder Aerosols for Pulmonary Protein Delivery.

PURPOSE: Pulmonary delivery of biologics is of great interest, as it can be used for the local treatment of respiratory diseases or as a route to systemic drug delivery. To reach the full potential of inhaled biologics, a formulation platform capable of producing high performance aerosols without altering protein native structure is required. METHODS: A formulation strategy using Particle Replication in Non-wetting Templates (PRINT) was developed to produce protein dry powders with precisely engineered particle morphology. Stability of the incorporated proteins was characterized and the aerosol properties of the protein dry powders was evaluated in vitro with an Andersen Cascade Impactor (ACI). RESULTS: Model proteins bovine serum albumin (BSA) and lysozyme were micromolded into 1 µm cylinders composed of more than 80% protein, by mass. Extensive characterization of the incorporated proteins found no evidence of alteration of native structures. The BSA formulation produced a mass median aerodynamic diameter (MMAD) of 1.77 µm ± 0.06 and a geometric standard deviation (GSD) of 1.51 ± 0.06 while the lysozyme formulation had an MMAD of 1.83 µm ± 0.12 and a GSD of 1.44 ± 0.03. CONCLUSION: Protein dry powders manufactured with PRINT could enable high-performance delivery of protein therapeutics to the lungs.

Mediating Passive Tumor Accumulation through Particle Size, Tumor Type, and Location.

As the enhanced permeation and retention (EPR) effect continues to be a controversial topic in nanomedicine, we sought to examine EPR as a function of nanoparticle size, tumor model, and tumor location, while also evaluating tumors for EPR mediating factors such as microvessel density, vascular permeability, lymphatics, stromal content, and tumor-associated immune cells. Tumor accumulation was evaluated for 55 × 60, 80 × 180, and 80 × 320 nm PRINT particles in four subcutaneous flank tumor models (SKOV3 human ovarian, 344SQ murine nonsmall cell lung, A549 human nonsmall cell lung, and A431 human epidermoid cancer). Each tumor model revealed specific particle accumulation trends with evident particle size dependence. Immuno-histochemistry staining revealed differences in tumor microvessel densities that correlated with overall tumor accumulation. Immunofluorescence images displayed size-mediated tumor penetration with signal from the larger particles concentrated close to the blood vessels, while signal from the smaller particle was observed throughout the tissue. Differences were also observed for the 55 × 60 nm particle tumor penetration across flank tumor models as a function of stromal content. The 55 × 60 nm particles were further evaluated in three orthotopic, metastatic tumor models (344SQ, A549, and SKOV3), revealing preferential accumulation in primary tumors and metastases over healthy tissue. Moreover, we observed higher tumor accumulation in the orthotopic lung cancer models than in the flank lung cancer models, whereas tumor accumulation was constant for both orthotopic and flank ovarian cancer models, further demonstrating the variability in the EPR effect as a function of tumor model and location.

The Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor Targeting of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells in vitro, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones in vivo. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice.

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.

Pulmonary Delivery of Butyrylcholinesterase as a Model Protein to the Lung.

Pulmonary delivery has great potential for delivering biologics to the lung if the challenges of maintaining activity, stability, and ideal aerosol characteristics can be overcome. To study the interactions of a biologic in the lung, we chose butyrylcholinesterase (BuChE) as our model enzyme, which has application for use as a bioscavenger protecting against organophosphate exposure or for use with pseudocholinesterase deficient patients. In mice, orotracheal administration of free BuChE resulted in 72 h detection in the lungs and 48 h in the broncheoalveolar lavage fluid (BALF). Free BuChE administered to the lung of all mouse backgrounds (Nude, C57BL/6, and BALB/c) showed evidence of an acute cytokine (IL-6, TNF-α, MIP2, and KC) and cellular immune response that subsided within 48 h, indicating relatively safe administration of this non-native biologic. We then developed a formulation of BuChE using Particle Replication in Non-Wetting Templates (PRINT). Aerosol characterization demonstrated biologically active BuChE 1 µm cylindrical particles with a mass median aerodynamic diameter of 2.77 µm, indicative of promising airway deposition via dry powder inhalers (DPI). Furthermore, particulate BuChE delivered via dry powder insufflation showed residence time of 48 h in the lungs and BALF. The in vivo residence time, immune response, and safety of particulate BuChE delivered via a pulmonary route, along with the cascade impaction distribution of dry powder PRINT BuChE, showed promise in the ability to deliver active enzymes with ideal deposition characteristics. These findings provide evidence for the feasibility of optimizing the use of BuChE in the clinic; PRINT BuChE particles can be readily formulated for use in DPIs, providing a convenient and effective treatment option.

Nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells.

Engineered nanoparticles have the potential to expand the breadth of pulmonary therapeutics, especially as respiratory vaccines. Notably, cationic nanoparticles have been demonstrated to produce superior local immune responses following pulmonary delivery; however, the cellular mechanisms of this increased response remain unknown. To this end, we investigated the cellular response of lung APCs following pulmonary instillation of anionic and cationic charged nanoparticles. While nanoparticles of both surface charges were capable of trafficking to the draining lymph node and were readily internalized by alveolar macrophages, both CD11b and CD103 lung dendritic cell (DC) subtypes preferentially associated with cationic nanoparticles. Instillation of cationic nanoparticles resulted in the upregulation of Ccl2 and Cxc10, which likely contributes to the recruitment of CD11b DCs to the lung. In total, these cellular mechanisms explain the increased efficacy of cationic formulations as a pulmonary vaccine carrier and provide critical benchmarks in the design of pulmonary vaccine nanoparticles. FROM THE CLINICAL EDITOR: Advance in nanotechnology has allowed the production of precise nanoparticles as vaccines. In this regard, pulmonary delivery has the most potential. In this article, the authors investigated the interaction of nanoparticles with various types of lung antigen presenting cells in an attempt to understand the cellular mechanisms. The findings would further help the future design of much improved vaccines for clinical use.

Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation.

In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle. However, in vivo, both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density. Overall, nanoparticle size and passive accumulation were the dominant factors eliciting tumor sequestration.

Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo.

To achieve the great potential of siRNA based gene therapy, safe and efficient systemic delivery in vivo is essential. Here we report reductively responsive hydrogel nanoparticles with highly uniform size and shape for systemic siRNA delivery in vivo. "Blank" hydrogel nanoparticles with high aspect ratio were prepared using continuous particle fabrication based on PRINT (particle replication in nonwetting templates). Subsequently, siRNA was conjugated to "blank" nanoparticles via a disulfide linker with a high loading ratio of up to 18 wt %, followed by surface modification to enhance transfection. This fabrication process could be easily scaled up to prepare large quantity of hydrogel nanoparticles. By controlling hydrogel composition, surface modification, and siRNA loading ratio, siRNA conjugated nanoparticles were highly tunable to achieve high transfection efficiency in vitro. FVII-siRNA conjugated nanoparticles were further stabilized with surface coating for in vivo siRNA delivery to liver hepatocytes, and successful gene silencing was demonstrated at both mRNA and protein levels.

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.

Preparation and biological evaluation of synthetic and polymer-encapsulated congeners of the antitumor agent pactamycin: insight into functional group effects and biological activity.

The synthesis and biological analysis of a number of novel congeners of the aminocyclopentitol pactamycin is described. Specific attention was paid to the preparation of derivatives at crucial synthetic branch points of the parent structure, and biological assays revealed a number of insights into the source of pactamycin's biological activity. Additionally, the encapsulation of pactamycin and select derivatives into the PRINT© nanoparticle technology was investigated as a proof-of-concept, and evidence of bioactivity modulation through nanoparticle delivery is demonstrated. This work has provided heretofore unrealized access to a large number of novel compounds for further evaluation.

Particle replication in nonwetting templates nanoparticles with tumor selective alkyl silyl ether docetaxel prodrug reduces toxicity.

Delivery systems designed to have triggered release after passively targeting the tumor may improve small molecule chemotherapeutic delivery. Particle replication in nonwetting templates was used to prepare nanoparticles to passively target solid tumors in an A549 subcutaneous xenograft model. An acid labile prodrug was delivered to minimize systemic free docetaxel concentrations and improve tolerability without compromising efficacy.

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.

RNA replicon delivery via lipid-complexed PRINT protein particles.

Herein we report the development of a nonviral lipid-complexed PRINT (particle replication in nonwetting templates) protein particle system (LPP particle) for RNA replicon delivery with a view toward RNA replicon-based vaccination. Cylindrical bovine serum albumin (BSA) particles (diameter (d) 1 µm, height (h) 1 µm) loaded with RNA replicon and stabilized with a fully reversible disulfide cross-linker were fabricated using PRINT technology. Highly efficient delivery of the particles to Vero cells was achieved by complexing particles with a mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids. Our data suggest that (1) this lipid-complexed protein particle is a promising system for delivery of RNA replicon-based vaccines and (2) it is necessary to use a degradable cross-linker for successful delivery of RNA replicon via protein-based particles.

Plasma, tumor and tissue pharmacokinetics of Docetaxel delivered via nanoparticles of different sizes and shapes in mice bearing SKOV-3 human ovarian carcinoma xenograft.

The particle fabrication technique PRINT® was used to fabricate monodisperse size and shape specific poly(lactide-co-glycolide) particles loaded with the chemotherapeutic Docetaxel. The pharmacokinetics of two cylindrical shaped particles with diameter=80nm; height=320nm (PRINT-Doc-80×320) and d=200nm; h=200nm (PRINT-Doc-200×200) were compared to Docetaxel in mice bearing human ovarian carcinoma SKOV-3 flank xenografts. The Docetaxel plasma exposure was ~20-fold higher for both particles compared to docetaxel. Additionally, the volume of distribution (Vd) of Docetaxel in PRINT formulations was ~18-fold (PRINT-Doc-80×320) and ~33-fold (PRINT-Doc-200×200) lower than Docetaxel. The prolonged duration of Docetaxel in plasma when dosed with PRINT formulations subsequently led to increased tumor exposure of Docetaxel from 0 to 168h (~53% higher for PRINT-Doc-80×320 and ~76% higher for PRINT-Doc-200×200 particles). PRINT-Doc-80×320 had lower exposures in the liver, spleen and lung compared with PRINT-Doc-200×200. Thus, the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system. FROM THE CLINICAL EDITOR: In this study, the plasma, tumor, and tissue pharmacokinetics of different Docetaxel nanoparticles of precise shape and size were characterized in mice with human ovarian carcinoma xenograft. It is concluded that the use of particles with smaller feature size may be preferred to decrease clearance by organs of the mononuclear phagocyte system.

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.

Rendering protein-based particles transiently insoluble for therapeutic applications.

Herein, we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery.