Tumor Vascular Remodeling Affects Molecular Dissemination to Lymph Node and Systemic Leukocytes.

Angiogenic and lymphangiogenic remodeling has long been accepted as a hallmark of cancer development and progression; however, the impacts of this remodeling on immunological responses, which are paramount to the responses to immunotherapeutic treatments, are underexplored. As immunotherapies represent one of the most promising new classes of cancer therapy, in this study, we explore the effects of angiogenic and lymphangiogenic normalization on dissemination of molecules injected into the tumor microenvironment to immune cells in lymph nodes draining the tumor as well as in systemically distributed tissues. A system of fluorescent tracers, size-matched to biomolecules of interest, was implemented to track different mechanisms of tumor transport and access to immune cells. This revealed that the presence of a tumor, and either angiogenic or lymphangiogenic remodeling, altered local retention of model biomolecules, trended toward normalizing dissemination to systemic organs, and modified access to lymph node-resident immune cells in manners dependent on mechanism of transport. More specifically, active cell migration by skin-derived antigen presenting cells was enhanced by both the presence of a tumor and lymphangiogenic normalization, while both angiogenic and lymphangiogenic normalization restored patterns of immune cell access to passively draining species. As a whole, this work uncovers the potential ramifications of tumor-induced angiogenesis and lymphangiogenesis, along with impacts of interrogation into these pathways, on access of tumor-derived species to immune cells. Impact Statement Angiogenic and lymphangiogenic normalization strategies have been utilized clinically to interrogate tumor vasculature with some success. In the age of immunotherapy, the impacts of these therapeutic interventions on immune remodeling are unclear. This work utilizes mouse models of angiogenic and lymphangiogenic normalization, along with a system of fluorescently tagged tracers, to uncover the impacts of angiogenesis and lymphangiogenesis on access of tumor-derived species to immune cell subsets within various organs.

Characterization of Inflammatory and Fibrotic Encapsulation Responses of Implanted Materials with Bacterial Infection.

Medical device infections are costly, while preclinical assessment of antimicrobial properties for new materials is time intensive and imperfect at capturing the interrelated aspects of infection response and wound resolution. Herein, we developed an in vivo model for quantification of inflammatory and biocompatibility responses in the presence of a sustained implant-associated infection. The antimicrobial effectiveness of commercially available polymer materials was compared to that of thermoplastic polyurethane (TPU) materials modified with putative antimicrobial strategies as example test materials. Materials were incubated with bioluminescent Escherichia coli prior to implantation in a dorsal subcutaneous pocket in rats with an additional intraluminal bolus of bacteria. Infection kinetics were monitored with bioluminescence, and inflammatory infiltrate and fibrous capsule thickness were determined from stained histological sections. Our model resulted in a persistent infection, sensitive to antimicrobial effects, as the materials modified with a putative antimicrobial surface were able to significantly reduce the level of infection in animals at day 4 postimplantation with efficacy similar to that of commercially available antimicrobial drug-eluting polymers (positive controls). At day 30 postimplantation, the antimicrobial surface modified TPU tubing was found to promote complete elimination of intraluminal bacteria in the absence of antibiotics. Differences were also measurable in acute inflammation, as Wright-Giemsa staining demonstrated reduced inflammatory cell infiltration at day 4 postimplantation for antimicrobial TPU materials. Additionally, antimicrobial materials exhibited reduced fibrous capsule thickness coinciding with infection resolution, as compared to unmodified TPU controls. The developed model can be utilized for testing antimicrobial polymers in the context of a prolonged infection while also revealing concurrent differences in the infiltrating immune cell profiles and fibrous capsule thickness, thus improving the relevance of preclinical medical device material testing.

Quantitation of lymphatic transport mechanism and barrier influences on lymph node-resident leukocyte access to lymph-borne macromolecules and drug delivery systems.

Lymph nodes (LNs) are tissues of the immune system that house leukocytes, making them targets of interest for a variety of therapeutic immunomodulation applications. However, achieving accumulation of a therapeutic in the LN does not guarantee equal access to all leukocyte subsets. LNs are structured to enable sampling of lymph draining from peripheral tissues in a highly spatiotemporally regulated fashion in order to facilitate optimal adaptive immune responses. This structure results in restricted nanoscale drug delivery carrier access to specific leukocyte targets within the LN parenchyma. Herein, a framework is presented to assess the manner in which lymph-derived macromolecules and particles are sampled in the LN to reveal new insights into how therapeutic strategies or drug delivery systems may be designed to improve access to dLN-resident leukocytes. This summary analysis of previous reports from our group assesses model nanoscale fluorescent tracer association with various leukocyte populations across relevant time periods post administration, studies the effects of bioactive molecule NO on access of lymph-borne solutes to dLN leukocytes, and illustrates the benefits to leukocyte access afforded by lymphatic-targeted multistage drug delivery systems. Results reveal trends consistent with the consensus view of how lymph is sampled by LN leukocytes resulting from tissue structural barriers that regulate inter-LN transport and demonstrate how novel, engineered delivery systems may be designed to overcome these barriers to unlock the therapeutic potential of LN-resident cells as drug delivery targets.

Polymer Microparticles Prolong Delivery of the 15-PGDH Inhibitor SW033291.

As the prevalence of age-related fibrotic diseases continues to increase, novel antifibrotic therapies are emerging to address clinical needs. However, many novel therapeutics for managing chronic fibrosis are small-molecule drugs that require frequent dosing to attain effective concentrations. Although bolus parenteral administrations have become standard clinical practice, an extended delivery platform would achieve steady-state concentrations over a longer time period with fewer administrations. This study lays the foundation for the development of a sustained release platform for the delivery of (+)SW033291, a potent, small-molecule inhibitor of the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) enzyme, which has previously demonstrated efficacy in a murine model of pulmonary fibrosis. Herein, we leverage fine-tuned cyclodextrin microparticles-specifically, ß-CD microparticles (ß-CD MPs)-to extend the delivery of the 15-PGDH inhibitor, (+)SW033291, to over one week.

Affinity-Based Polymers Provide Long-Term Immunotherapeutic Drug Delivery Across Particle Size Ranges Optimal for Macrophage Targeting.

Drug delivery to specific arms of the immune system can be technically challenging to provide prolonged drug release while limiting off-target toxicity given the limitations of current drug delivery systems. In this work, we test the design of a cyclodextrin (CD) polymer platform to extend immunomodulatory drug delivery via affinity interactions for sustained release at multiple size scales. The parameter space of synthesis variables influencing particle nucleation and growth (pre-incubation time and stirring speed) and post-synthesis grinding effects on resulting particle diameter were characterized. We demonstrate that polymerized CD forms exhibit size-independent release profiles of the small molecule drug lenalidomide (LND) and can provide improved drug delivery profiles versus macro-scale CD polymer disks in part due to increased loading efficiency. CD polymer microparticles and smaller, ground particles demonstrated no significant cytotoxicity as compared to the base CD monomer when co-incubated with fibroblasts. Uptake of ground CD particles was significantly higher following incubation with RAW 264.7 macrophages in culture over standard CD microparticles. Thus, the affinity/structure properties afforded by polymerized CD allow particle size to be modified to affect cellular uptake profiles independently of drug release rate for applications in cell-targeted drug delivery.

Affinity Effects on the Release of Non-Conventional Antifibrotics from Polymer Depots.

For many chronic fibrotic conditions, there is a need for local, sustained antifibrotic drug delivery. A recent trend in the pharmaceutical industry is the repurposing of approved drugs. This paper investigates drugs that are classically used for anthelmintic activity (pyrvinium pamoate (PYR)), inhibition of adrenal steroidgenesis (metyrapone (MTP)), bactericidal effect (rifampicin (RIF), and treating iron/aluminum toxicity (deferoxamine mesylate (DFOA)), but are also under investigation for their potential positive effect in wound healing. In this role, they have not previously been tested in a localized delivery system suitable for obtaining the release for the weeks-to-months timecourse needed for wound resolution. Herein, two cyclodextrin-based polymer systems, disks and microparticles, are demonstrated to provide the long-term release of all four tested non-conventional wound-healing drugs for up to 30 days. Higher drug affinity binding, as determined from PyRx binding simulations and surface plasmon resonance in vitro, corresponded with extended release amounts, while drug molecular weight and solubility correlated with the improved drug loading efficiency of cyclodextrin polymers. These results, combined, demonstrate that leveraging affinity interactions, in combination with drug choice, can extend the sustained release of drugs with an alternative, complimentary action to resolve wound-healing and reduce fibrotic processes.

Serum biomolecules unable to compete with drug refilling into cyclodextrin polymers regardless of the form.

Polymers that are refillable and sustain local release will have a great impact in both preventing and treating local cancer recurrence as well as addressing non-resectable diseases. Polymerized cyclodextrin (pCD) disks, which reload drugs into molecular "pockets" in vivo through affinity interactions, have been previously shown to localize doxorubicin (Dox) to treat glioblastoma multiforme. However, one concern is whether drug refilling is influenced by competition from local biomolecules. In addition the impact of the polymer form on drug refilling is unknown. Herein, different pCD formulations were synthesized from γ-cyclodextrin (γ-CD) and were compared in vitro using competitive drug filling/refilling assays. Data reveal that affinity-based drug refilling occurs as a function of both the polymer form and the sustained release polymeric liquid (SRPL) dilution factor, pointing to the surface/volume ratio, as well as the CD pocket density, and the effects of the distance between pocket. In vitro refilling experiments with cholesterol demonstrated no interference with Dox filling of the CD polymer, while the presence of albumin only slightly reduced Dox filling of pCD-γ-MP (microparticle) and pCD-γ-SRPL forms, but not pCD-γ-disks. Moreover, whole serum competition did not inhibit filling or refilling of pCD-γ-MP with Dox at multiple concentrations and filling times, which indicates that this polymer (re)filling is primarily driven by affinity-based interactions that can overcome the physiological conditions which may limit other drug delivery approaches. This was supplemented by isolating variables through docking simulations and affinity measurements. These results attest to the efficiency of in vivo or in situ polymer filling/refilling in the presence of competitive biological molecules achieved partially through high affinity drug to polymer interactions.

Cyclodextrin Polymer Preserves Sirolimus Activity and Local Persistence for Antifibrotic Delivery over the Time Course of Wound Healing.

Fibrosis and dysphagic stricture of the esophagus is a major unaddressed problem often accompanying endoscopic removal of esophageal cancers and precancerous lesions. While weekly injections of antiproliferative agents show potential for improved healing, repeated injections are unlikely clinically and may alternatively be replaced by creating an esophageal drug delivery system. Affinity-based polymers have previously shown success for continuous delivery of small molecules for weeks to months. Herein, we explored the potential of an affinity-based microparticle to provide long-term release of an antiproliferative drug, sirolimus. In molecular docking simulations and surface plasmon resonance experiments, sirolimus was found to have suitable affinity for beta-cyclodextrin, while dextran, as a low affinity control, was validated. Polymerized beta-cyclodextrin microparticles exhibited 30 consecutive days of delivery of sirolimus during in vitro release studies. In total, the polymerized beta-cyclodextrin microparticles released 36.9 mg of sirolimus per milligram of polymer after one month of incubation in vitro. Taking daily drug release aliquots and applying them to PT-K75 porcine mucosal fibroblasts, we observed that cyclodextrin microparticle delivery preserved bioactivity of sirolimus inhibiting proliferation by 27-67% and migration of fibroblasts by 28-100% of buffer treated controls in vitro. Testing for esophageal injection site losses, no significant loss was incurred under simulated saliva flow for 10 min, and 16.7% of fluorescently labeled polymerized cyclodextrin microparticle signal was retained at 28 days after submucosal injection in esophageal tissue ex vivo versus only 4% of the initial amount remaining for free dye molecules injected alone. By combining affinity-based drug delivery for continuous long-term release with a microparticle platform that is injectable yet remains localized in tissue interstitium, this combination platform demonstrates promise for preventing esophageal fibrosis and stricture.

Molecular Imprinting of Cyclodextrin Supramolecular Hydrogels Improves Drug Loading and Delivery.

Cyclodextrin-based controlled delivery materials have previously been developed for controlled release of different therapeutic drugs. In this study, a supramolecular hydrogel made from cyclodextrin-based macromonomers is subjected to molecular imprinting to investigate the impact on release kinetics and drug loading, when compared with non-imprinted, or alternately imprinted hydrogels. Mild synthesis conditions are used to molecularly imprint three antibiotics-novobiocin, rifampicin, and vancomycin-and to test two different hydrogel chemistries. The release profile and drug loading of the molecularly imprinted hydrogels are characterized using ultraviolet spectroscopy over a period of 35 days and compared to non-imprinted, and alternately imprinted hydrogels. While only modest differences are observed in the release rate of the antibiotics tested, a substantial difference is observed in the total drug-loading amount possible for hydrogels releasing drugs which has been templated by those drugs. Hydrogels releasing drugs which are templated by other drugs do not show improved release or loading. Analysis by FTIR does not show substantial incorporation of drug into the polymer. Lastly, bioactivity assays confirmed long-term stability and release of incorporated antibiotics.

Multifunctional decoration of alpha-tocopheryl succinate-based NP for cancer treatment: effect of TPP and LTVSPWY peptide.

Active targeting not only of a specific cell but also a specific organelle maximizes the therapeutic activity minimizing adverse side effects in healthy tissues. The present work describes the synthesis, characterization, and in vitro biological activity of active targeting nanoparticles (NP) for cancer therapy based on α-tocopheryl succinate (α-TOS), a well-known mitocan, that selectively induces apoptosis of cancer cells and proliferalting endothelial cells. Human epidermal growth factor receptor 2 (HER2) targeting peptide LTVSPWY (PEP) and triphenylphosphonium lipophilic cation (TPP) were conjugated to a previously optimized RAFT block copolymer that formed self-assembled NP of appropriate size for this application and low polydispersity by self-organized precipitation method. PEP and TPP were included in order to target not only HER2 positive cancer cells, but also the mitochondria of these cancer cells, respectively. The in vitro experiments demonstrated the faster incorporation of the active-targeting NP and the higher accumulation of TPP-bearing NP in the mitochondria of MDA-MB-453 HER2 positive cancer cells compared to non-decorated NP. Moreover, the encapsulation of additional α-TOS in the hydrophobic core of the NP was achieved with high efficiencies. The loaded NP presented higher cytotoxicity than unloaded NP but preserved their selectivity against cancer cells in a range of tested concentrations.

Photothermal and photodynamic activity of polymeric nanoparticles based on α-tocopheryl succinate-RAFT block copolymers conjugated to IR-780.

The aim of this work was the generation of a multifunctional nanopolymeric system that incorporates IR-780 dye, a near-infrared (NIR) imaging probe that exhibits photothermal and photodynamic properties; and a derivate of α-tocopheryl succinate (α-TOS), a mitochondria-targeted anticancer compound. IR-780 was conjugated to the hydrophilic segment of copolymer PEG-b-polyMTOS, based on poly(ethylene glycol) (PEG) and a methacrylic derivative of α-tocopheryl succinate (MTOS), to generate IR-NP, self-assembled nanoparticles (NPs) in aqueous media which exhibit a hydrophilic shell and a hydrophobic core. During assembly, the hydrophobic core of IR-NP could encapsulate additional IR-780 to generate derived subspecies carrying different amount of probe (IR-NP-eIR). Evaluation of photo-inducible properties of IR-NP and IR-NP-eIR were thoroughly assessed in vitro. Developed nanotheranostic particles showed distinct fluorescence and photothermal behavior after excitation by a laser light emitting at 808nm. Treatment of MDA-MB-453 cells with IR-NP or IR-NP-eIR resulted in an efficient internalization of the IR-780 dye, while subsequent NIR-laser irradiation led to a severe decrease in cell viability. Photocytoxicity conducted by IR-NP, which could not be attributed to the generation of lethal hyperthermia, responded to an increase in the levels of intracellular reactive oxygen species (ROS). Therefore, the fluorescence imaging and inducible phototoxicity capabilities of NPs derived from IR-780-PEG-b-polyMTOS copolymer confer high value to these nanotheranostics tools in clinical cancer research. STATEMENT OF SIGNIFICANCE: Multifunctional polymeric nanoparticles (NPs) that combine imaging and therapeutic properties are highly valuable in cancer treatment. In this paper we describe the development of NPs that are fluorescent in the near-infrared (NIR). This is important for their visualization in living tissues that present low absorption and low autofluorescence in this wavelength region (between 700 and 1000nm). Moreover, NPs present photothermal and photodynamic properties when NIR irradiated: the NPs produce an efficient increment of temperature and increase the intracellular reactive oxygen species (ROS) when laser irradiated at 808nm. These tuneable photoinduced properties make the NPs highly cytotoxic after NIR irradiation and provide a new tool for highly precise cancer treatment.

Enhanced Bioactivity of α-Tocopheryl Succinate Based Block Copolymer Nanoparticles by Reduced Hydrophobicity.

Well-structured amphiphilic copolymers are necessary to obtain self-assembled nanoparticles (NPs) based on synthetic polymers. Highly homogeneous and monodispersed macromolecules obtained by controlled polymerization have successfully been used for this purpose. However, disaggregation of the organized macromolecules is desired when a bioactive element, such as α-tocopheryl succinate, is introduced in self-assembled NPs and this element must be exposed or released to exert its action. The aim of this work is to demonstrate that the bioactivity of synthetic NPs based on defined reversible addition-fragmentation chain transfer polymerization copolymers can be enhanced by the introduction of hydrophilic comonomers in the hydrophobic segment. The amphiphilic terpolymers are based on poly(ethylene glycol) (PEG) as hydrophilic block, and a hydrophobic block based on a methacrylic derivative of α-tocopheryl succinate (MTOS) and small amounts of 2-hydroxyethyl methacrylate (HEMA) (PEG-b-poly(MTOS-co-HEMA)). The introduction of HEMA reduces hydrophobicity and introduces "disorder" both in the homogeneous blocks and the compact core of the corresponding NPs. These NPs are able to encapsulate additional α-tocopheryl succinate (α-TOS) with high efficiency and their biological activity is much higher than that described for the unmodified copolymers, proposedly due to more efficient degradation and release of α-TOS, demonstrating the importance of the hydrophilic-hydrophobic balance.

Implications of Lymphatic Transport to Lymph Nodes in Immunity and Immunotherapy.

Adaptive immune response consists of many highly regulated, multistep cascades that protect against infection while preserving the health of autologous tissue. The proper initiation, maintenance, and resolution of such responses require the precise coordination of molecular and cellular signaling over multiple time and length scales orchestrated by lymphatic transport. In order to investigate these functions and manipulate them for therapy, a comprehensive understanding of how lymphatics influence immune physiology is needed. This review presents the current mechanistic understanding of the role of the lymphatic vasculature in regulating biomolecule and cellular transport from the interstitium, peripheral tissue immune surveillance, the lymph node stroma and microvasculature, and circulating lymphocyte homing to lymph nodes. This review also discusses the ramifications of lymphatic transport in immunity as well as tolerance and concludes with examples of how lymphatic-mediated targeting of lymph nodes has been exploited for immunotherapy applications.