Nanotargeted Delivery of Immune Therapeutics in Type 1 Diabetes.

Immune therapeutics holds great promise in the treatment of type 1 diabetes (T1D). Nonetheless, their progress is hampered by limited efficacy, equipoise, or issues of safety. To address this, a novel and specific nanodelivery platform for T1D that targets high endothelial venules (HEVs) presented in the pancreatic lymph nodes (PLNs) and pancreas is developed. Data indicate that the pancreata of nonobese diabetic (NOD) mice and patients with T1D are unique in their expression of newly formed HEVs. Anti-CD3 mAb is encapsulated in poly(lactic-co-glycolic acid)-poly(ethylene glycol) nanoparticles (NPs), the surfaces of which are conjugated with MECA79 mAb that recognizes HEVs. Targeted delivery of these NPs improves accumulation of anti-CD3 mAb in both the PLNs and pancreata of NOD mice. Treatment of hyperglycemic NOD mice with MECA79-anti-CD3-NPs results in significant reversal of T1D compared to those that are untreated, treated with empty NPs, or provided free anti-CD3. This effect is associated with a significant reduction of T effector cell populations in the PLNs and a decreased production of pro-inflammatory cytokine in the mice treated with MECA79-anti-CD3-NPs. In summary, HEV-targeted therapeutics may be used as a means by which immune therapeutics can be delivered to PLNs and pancreata to suppress autoimmune diabetes effectively.

Characterization of CD41+ cells in the lymph node.

Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.

Intratumoral immunotherapy using platelet-cloaked nanoparticles enhances antitumor immunity in solid tumors.

Intratumoral immunotherapy is an emerging modality for the treatment of solid tumors. Toll-like receptor (TLR) agonists have shown promise for eliciting immune responses, but systemic administration often results in the development of adverse side effects. Herein, we investigate whether localized delivery of the TLR agonist, resiquimod (R848), via platelet membrane-coated nanoparticles (PNP-R848) elicits antitumor responses. The membrane coating provides a means of enhancing interactions with the tumor microenvironment, thereby maximizing the activity of R848. Intratumoral administration of PNP-R848 strongly enhances local immune activation and leads to complete tumor regression in a colorectal tumor model, while providing protection against repeated tumor re-challenges. Moreover, treatment of an aggressive breast cancer model with intratumoral PNP-R848 delays tumor growth and inhibits lung metastasis. Our findings highlight the promise of locally delivering immunostimulatory payloads using biomimetic nanocarriers, which possess advantages such as enhanced biocompatibility and natural targeting affinities.

Selective Trafficking of Light Chain-Conjugated Nanoparticles to the Kidney and Renal Cell Carcinoma.

Specific delivery platforms for drugs to the kidney and diagnostic agents to renal cell carcinoma (RCC) constitute urgent but unfulfilled clinical needs. To address these challenges, we engineered nanocarriers that interact selectively for the first time with proximal tubule epithelial cells (PTECs) in the kidney and with RCC through the interplay between lambda light chains (LCs) attached to PEGylated polylactic-co-glycolic acid (PLGA) nanoparticles and the membrane protein megalin. Systemic administration of these light chain-conjugated nanoparticles (LC-NPs) to mice resulted in their specific retention by megalin-expressing PTECs for seven days. Repetitive dosing of LC-NPs demonstrated no renal toxicity. LC-NPs also localized selectively to megalin-expressing RCC tumors in mice. Moreover, we confirmed that both the primary tumor and lymph node metastases of human RCC express megalin, reinforcing the potential of LC-NPs for clinical use. Thus, LC-NPs can contribute potentially to improving the management of both non-oncologic and oncologic renal disorders.

Nanodelivery of Mycophenolate Mofetil to the Organ Improves Transplant Vasculopathy.

Inflammation occurring within the transplanted organ from the time of harvest is an important stimulus of early alloimmune reactivity and promotes chronic allograft rejection. Chronic immune-mediated injury remains the primary obstacle to the long-term success of organ transplantation. However, organ transplantation represents a rare clinical setting in which the organ is accessible ex vivo, providing an opportunity to use nanotechnology to deliver therapeutics directly to the graft. This approach facilitates the directed delivery of immunosuppressive agents (ISA) to target local pathogenic immune responses prior to the transplantation. Here, we have developed a system of direct delivery and sustained release of mycophenolate mofetil (MMF) to treat the donor organ prior to transplantation. Perfusion of a donor mouse heart with MMF-loaded PEG-PLGA nanoparticles (MMF-NPs) prior to transplantation abrogated cardiac transplant vasculopathy by suppressing intragraft pro-inflammatory cytokines and chemokines. Our findings demonstrate that ex vivo delivery of an ISA to donor organs using a nanocarrier can serve as a clinically feasible approach to reduce transplant immunity.

Ectopic high endothelial venules in pancreatic ductal adenocarcinoma: A unique site for targeted delivery.

BACKGROUND: Nanomedicine offers an excellent opportunity to tackle treatment-refractory malignancies by enhancing the delivery of therapeutics to the tumor site. High endothelial venules (HEVs) are found primarily in lymph nodes or formed de novo in peripheral tissues during inflammatory responses. They express peripheral node addressin (PNAd), which is recognized by the monoclonal antibody MECA79. METHODS: Here, we demonstrated that HEVs form de novo in human pancreatic ductal adenocarcinoma (PDAC). We engineered MECA79 coated nanoparticles (MECA79-NPs) that recognize these ectopic HEVs in PDAC. FINDINGS: The trafficking of MECA79-NPs following intravenous delivery to human PDAC implanted in a humanized mouse model was more robust than non-conjugated NPs. Treatment with MECA79-Taxol-NPs augmented the delivery of Paclitaxel (Taxol) to the tumor site and significantly reduced the tumor size. This effect was associated with a higher apoptosis rate of PDAC cells and reduced vascularization within the tumor. INTERPRETATION: Targeting the HEVs of PDAC using MECA79-NPs could lay the ground for the localized delivery of a wide variety of drugs including chemotherapeutic agents. FUND: National Institutes of Health (NIH) grants: T32-EB016652 (B·B.), NIH Cancer Core Grant CA034196 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.).

Targeting antigen-presenting cells by anti-PD-1 nanoparticles augments antitumor immunity.

Recent studies in cancer research have focused intensely on the antineoplastic effects of immune checkpoint inhibitors. While the development of these inhibitors has progressed successfully, strategies to further improve their efficacy and reduce their toxicity are still needed. We hypothesized that the delivery of anti-PD-1 antibody encapsulated in PLGA nanoparticles (anti-PD-1 NPs) to the spleen would improve the antitumor effect of this agent. Unexpectedly, we found that mice treated with a high dose of anti-PD-1 NPs exhibited significantly higher mortality compared with those treated with free anti-PD-1 antibody, due to the overactivation of T cells. Administration of anti-PD-1 NPs to splenectomized LT-α-/- mice, which lack both lymph nodes and spleen, resulted in a complete reversal of this increased mortality and revealed the importance of secondary lymphoid tissues in mediating anti-PD-1-associated toxicity. Attenuation of the anti-PD-1 NPs dosage prevented toxicity and significantly improved its antitumor effect in the B16-F10 murine melanoma model. Furthermore, we found that anti-PD-1 NPs undergo internalization by DCs in the spleen, leading to their maturation and the subsequent activation of T cells. Our findings provide important clues that can lead to the development of strategies to enhance the efficacy of immune checkpoint inhibitors.

Targeted delivery of immune therapeutics to lymph nodes prolongs cardiac allograft survival.

The targeted delivery of therapeutic drugs to lymph nodes (LNs) provides an unprecedented opportunity to improve the outcomes of transplantation and immune-mediated diseases. The high endothelial venule is a specialized segment of LN vasculature that uniquely expresses peripheral node addressin (PNAd) molecules. PNAd is recognized by MECA79 mAb. We previously generated a MECA79 mAb-coated microparticle (MP) that carries tacrolimus. Although this MP trafficked to LNs, it demonstrated limited therapeutic efficacy in our transplant model. Here, we have synthesized a nanoparticle (NP) as a carrier of anti-CD3, and optimized the conjugation strategy to coat the NP surface with MECA79 mAb (MECA79-anti-CD3-NP) to enhance LN accumulation. As compared with nonconjugated NPs, a significantly higher quantity of MECA79-NPs accumulated in the draining lymph node (DLN). Many MECA79-NPs underwent internalization by T cells and dendritic cells within the LNs. Short-term treatment of murine cardiac allograft recipients with MECA79-anti-CD3-NP resulted in significantly prolonged allograft survival in comparison with the control groups. Prolonged graft survival following treatment with MECA79-anti-CD3-NP was characterized by a significant increase in intragraft and DLN Treg populations. Treg depletion abrogated the prolongation of heart allograft survival. We believe this targeted approach of drug delivery could redefine the methods of administering immune therapeutics in transplantation.

Repetitive ischemic injuries to the kidneys result in lymph node fibrosis and impaired healing.

The contribution of the kidney-draining lymph node (KLN) to the pathogenesis of ischemia-reperfusion injury (IRI) of the kidney and its subsequent recovery has not been explored in depth. In addition, the mechanism by which repetitive IRI contributes to renal fibrosis remains poorly understood. Herein, we have found that IRI of the kidney is associated with expansion of high endothelial venules (HEVs) and activation of fibroblastic reticular cells (FRCs) in the KLN, as demonstrated by significant expansion in the extracellular matrix. The lymphotoxin α signaling pathway mediates activation of FRCs, and chronic treatment with lymphotoxin ß receptor-immunoglobulin fusion protein (LTßr-Ig) resulted in marked alteration of the KLN as well as augmentation of renal fibrosis. Depletion of FRCs reduced T cell activation in the KLN and ameliorated renal injury in acute IRI. Repetitive renal IRI was associated with senescence of FRCs, fibrosis of the KLN, and renal scarring, which were ameliorated by FRC administration. Therefore, our study emphasizes the critical role of FRCs in both the initiation and repair phases of injury following IRI of the kidney.

Active targeted delivery of immune therapeutics to lymph nodes.

PURPOSE OF REVIEW: Organ transplantation is a life-saving procedure and the only option for patients with end-organ failure. Immune therapeutics have been key to the success of organ transplantation. However, immune therapeutics are still unable to eliminate graft rejection and their toxicity has been implicated in poorer long-term transplant outcomes. Targeted nanodelivery has the potential to enhance not only the therapeutic index but also the bioavailability of the immune therapeutics. One of the key sites of immune therapeutics delivery is lymph node where the priming of immune cells occur. The focus of this review is on nanomedicine research to develop the targeted delivery of immune therapeutics to lymph nodes for controlling immune activation. RECENT FINDINGS: As nanomedicine creates its niche in clinical care, it provides novel immunotherapy platforms for transplant recipients. Draining lymph nodes are the primary loci of immune activation and represent a formidable site for delivery of wide variety of immune therapeutics. There have been relentless efforts to improve the properties of nanomedicines, to have in-depth knowledge of antigen and drug loading, and, finally, to explore various routes of passive and active targeted delivery to lymph nodes. SUMMARY: The application of nanotechnology principles in the delivery of immune therapeutics to the lymph node has created enormous excitement as a paradigm shifting approach that enables targeted delivery of a gamut of molecules to achieve a desired immune response. Therefore, innovative strategies that improve their efficacy while reducing their toxicity are among the highest unmet needs in transplantation.

Virus-resembling nano-structures for near infrared fluorescence imaging of ovarian cancer HER2 receptors.

Ovarian cancer remains the dominant cause of death due to malignancies of the female reproductive system. The capability to identify and remove all tumors during intraoperative procedures may ultimately reduce cancer recurrence, and lead to increased patient survival. The objective of this study is to investigate the effectiveness of an optical nano-structured system for targeted near infrared (NIR) imaging of ovarian cancer cells that over-express the human epidermal growth factor receptor 2 (HER2), an important biomarker associated with ovarian cancer. The nano-structured system is comprised of genome-depleted plant-infecting brome mosaic virus doped with NIR chromophore, indocyanine green, and functionalized at the surface by covalent attachment of monoclonal antibodies against the HER2 receptor. We use absorption and fluorescence spectroscopy, and dynamic light scattering to characterize the physical properties of the constructs. Using fluorescence imaging and flow cytometry, we demonstrate the effectiveness of these nano-structures for targeted NIR imaging of HER2 receptors in vitro. These functionalized nano-materials may provide a platform for NIR imaging of ovarian cancer.

Functionalized polymeric nanoparticles loaded with indocyanine green as theranostic materials for targeted molecular near infrared fluorescence imaging and photothermal destruction of ovarian cancer cells.

BACKGROUND AND OBJECTIVES: Ovarian cancer remains the deadliest malignancy of the female reproductive system. The ability to identify and destroy all ovarian tumor nodules may have a termendous impact on preventing tumor recurrence, and patient survival. The objective of this study is to investigate the effectiveness of a nano-structured system for combined near infrared (NIR) fluorescence imaging of human epidermal growth factor receptor-2 (HER2) over-expression, as a biomarker of ovarian cancer cells, and photothermal destruction of these cells in vitro. MATERIALS AND METHODS: The nano-structured system consists of the near infrared dye, indocyanine green (ICG), encapsulated within poly(allylamine) hydrochloride chains cross-linked ionically with sodium phosphate. The surface of the construct is functionalized by covalently attached polyethylene glycol, and monoclonal antibodies against HER2 using reducitve amination methods. We use dynamic light scattering, and absorption and fluorescence spectroscopy for phyiscal characterization of the constructs. Flow cytometry and fluorescence microscopy are used to investigate molecular targeting and imaging capabilities of the constructs against SKOV3 and OVCAR3 ovarian cancer cell lines, which have relatively high and low expression levels of the HER2 receptor, respectively. Continuous NIR laser irradiation at 808 nm is used to investigating the utility of the constructs in mediating photothermal destruction of SKOV3 cells. RESULTS: Flow cytometry results indicate that the functionalized nano-constructs are more effective in targeting the HER2 receptor than non-encapsulated ICG and non-functionlaized constructs (P < 0.005). Fluorescence microscopic images show the capaiblity of the functionalized constructs in NIR imaging of HER2 overexpression. The functionalized nano-constructs are also capable of inducing a significantly greater increase in photothermal destruction of SKOV3 cells than free ICG and non-functionalized constructs (P < 0.005). CONCLUSION: We have demonstrated the efficacy of polymeric nano-structured constructs loaded with ICG, and functionalized with the monoclonal antibodies, as thernaostic materials for targted molecular NIR imaging of the HER2 receptor overexpression on ovarian cancer cells, and photothermal destruction of these cells. These nanoparticles may prove useful towards intraoperative detection, imaging, and phototherapy of small ovarian cancer nodules.

Erythrocyte-derived photo-theranostic agents: hybrid nano-vesicles containing indocyanine green for near infrared imaging and therapeutic applications.

Development of theranostic nano-constructs may enable diagnosis and treatment of diseases at high spatial resolution. Some key requirements for clinical translation of such constructs are that they must be non-toxic, non-immunogenic, biodegradable, with extended circulating lifetime. Cell-based structures, particularly those derived from erythrocytes, are promising candidate carrier systems to satisfy these requirements. One particular type of theranostic materials utilize light-sensitive agents that once photo-activated can provide diagnostic imaging capability, and elicit therapeutic effects. Here we demonstrate the first successful engineering of hybrid nano-scale constructs derived from membranes of hemoglobin-depleted erythrocytes that encapsulate the near infrared chromophore, indocyanine green. We show the utility of the constructs as photo-theranostic agents in fluorescence imaging and photothermal destruction of human cells. These erythrocyte-mimicking nano-structures can be derived autologously, and may have broad applications in personal nanomedicine ranging from imaging and photo-destruction of cancerous tissues to vascular abnormalities, and longitudinal evaluations of therapeutic interventions.

Effects of nanoencapsulation and PEGylation on biodistribution of indocyanine green in healthy mice: quantitative fluorescence imaging and analysis of organs.

Near-infrared nanoconstructs present a potentially effective platform for site-specific and deep tissue optical imaging and phototherapy. We have engineered a polymeric nanocapsule composed of polyallylamine hydrochloride (PAH) chains cross-linked with sodium phosphate and doped with indocyanine green (ICG) toward such endeavors. The ICG-doped nanocapsules were coated covalently with polyethylene glycol (5000 daltons) through reductive amination. We administrated the constructs by tail vein injection to healthy mice. To characterize the biodistribution of the constructs, we performed in vivo quantitative fluorescence imaging and subsequently analyzed the various extracted organs. Our results suggest that encapsulation of ICG in these PEGylated constructs is an effective approach to prolong the circulation time of ICG and delay its hepatic accumulation. Increased bioavailability of ICG, due to encapsulation, offers the potential of extending the clinical applications of ICG, which are currently limited due to rapid elimination of ICG from the vasculature. Our results also indicate that PAH and ICG-doped nanocapsules (ICG-NCs) are not cytotoxic at the levels used in this study.

Coatings of polyethylene glycol for suppressing adhesion between solid microspheres and flat surfaces.

This article describes the development and the examination of surface coatings that suppress the adhesion between glass surfaces and polymer microspheres. Superparamagnetic doping allowed for exerting magnetic forces on the microbeads. The carboxyl functionalization of the polymer provided the means for coating the beads with polyethylene glycol (PEG) with different molecular weight. Under gravitational force, the microbeads settled on glass surfaces with similar polymer coatings. We examined the efficacy of removing the beads from the glass surfaces by applying a pulling force of ~1.2 pN. The percent beads remaining on the surface after applying the pulling force for approximately 5 s served as an indication of the adhesion propensity. Coating of PEG with molecular weight ranging between 3 and 10 kDa was essential for suppressing the adhesion. For the particular substrates, surface chemistry and aqueous media we used, coatings of 5 kDa manifested optimal suppression of adhesion: that is, only 3% of the microbeads remained on the surface after applying the pulling magnetic force. When either the glass or the beads were not PEGylated, the adhesion between them was substantial. Addition of a noncharged surfactant, TWEEN, above its critical micelle concentrations (CMCs) suppressed the adhesion between noncoated substrates. The extent of this surfactant-induced improvement of the adhesion suppression, however, did not exceed the quality of preventing the adhesion that we attained by PEGylating both substrates. In addition, the use of surfactants did not significantly improve the suppression of bead-surface adhesion when both substrates were PEGylated. These findings suggest that such surfactant additives tend to be redundant and that covalently grafted coatings of PEGs with selected chain lengths provide sufficient suppression of nonspecific interfacial interactions.

Effect of polyethylene glycol coatings on uptake of indocyanine green loaded nanocapsules by human spleen macrophages in vitro.

Near-infrared (NIR) optically active nanoparticles are promising exogenous chromophores for applications in medical imaging and phototherapy. Since nanoparticles can be rapidly eliminated from the body by cells of the reticuloendothelial system, a thriving strategy to increase their blood circulation time is through surface modification with polyethylene glycol (PEG). We constructed polymeric nanocapsules loaded with indocyanine green (ICG), an FDA-approved NIR dye, and coated with aldehyde-terminated PEG. Using optical absorbance spectroscopy and flow cytometry, we investigated the effect of PEG coating and molecular weight (MW) of PEG [5000 and 30,000 Daltons (Da)] on the phagocytic content of human spleen macrophages incubated with ICG-containing nanocapsules (ICG-NCs) between 15 to 360 min. Our results indicate that surface coating with PEG is an effective method to reduce the phagocytic content of ICG-NCs within macrophages for at least up to 360 min of incubation time. Coating the surface of ICG-NCs with the low MW PEG results in lower phagocytic content of ICG-NCs within macrophages for at least up to 60 min of incubation time as compared to ICG-NCs coated with the high MW PEG. Surface coating of ICG-NCs with PEG is a promising approach to prolong vasculature circulation time of ICG for NIR imaging and phototherapeutic applications.

Kinetics of bacterial fluorescence staining with 3,3'-diethylthiacyanine.

For more than a century, colorimetric and fluorescence staining have been the foundation of a broad range of key bioanalytical techniques. The dynamics of such staining processes, however, still remains largely unexplored. We investigated the kinetics of fluorescence staining of two gram-negative and two gram-positive species with 3,3'-diethylthiacyanine (THIA) iodide. An increase in the THIA fluorescence quantum yield, induced by the bacterial dye uptake, was the principal reason for the observed emission enhancement. The fluorescence quantum yield of THIA depended on the media viscosity and not on the media polarity, which suggested that the microenvironment of the dye molecules taken up by the cells was restrictive. The kinetics of fluorescence staining did not manifest a statistically significant dependence neither on the dye concentration, nor on the cell count. In the presence of surfactant additives, however, the fluorescence-enhancement kinetic patterns manifested species specificity with statistically significant discernibility.