Nanocellulose is increasingly considered for applications; however, the fibrillar nature, crystalline phase, and surface reactivity of these high aspect ratio nanomaterials need to be considered for safe biomedical use. Here a comprehensive analysis of the impact of cellulose nanofibrils (CNF) and nanocrystals (CNC) is performed using materials provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences. An intermediary length of nanocrystals is also derived by acid hydrolysis. While all CNFs and CNCs are devoid of cytotoxicity, 210 and 280 nm fluorescein isothiocyanate (FITC)-labeled CNCs show higher cellular uptake than longer and shorter CNCs or CNFs. Moreover, CNCs in the 200-300 nm length scale are more likely to induce lysosomal damage, NLRP3 inflammasome activation, and IL-1β production than CNFs. The pro-inflammatory effects of CNCs are correlated with higher crystallinity index, surface hydroxyl density, and reactive oxygen species generation. In addition, CNFs and CNCs can induce maturation of bone marrow-derived dendritic cells and CNCs (and to a lesser extent CNFs) are found to exert adjuvant effects in ovalbumin (OVA)-injected mice, particularly for 210 and 280 nm CNCs. All considered, the data demonstrate the importance of length scale, crystallinity, and surface reactivity in shaping the innate immune response to nanocellulose.

While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We demonstrate a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway. This is accomplished by conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows prodrug self-assembly into nanovesicles or incorporation into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). The nanovesicles plus free OX or OX/IND-MSNP induce effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication is accomplishable by recruiting cytotoxic T lymphocytes, concomitant with downregulation of Foxp3⁺ T cells.

Polyrotaxane (PRX) is a promising supramolecular carrier for gene delivery. Classic PRX exhibits a linear structure in which the amine-functionalized α-cyclodextrin (CD) is threaded along the entire polyethylene glycol (PEG) backbone. While promising in vitro, the absence of free PEG moieties after CD threading compromised the in vivo implementation, due to the unfavorable pharmacokinetics (PK) and biodistribution profile. Herein, we developed a multi-arm PRX nanocarrier platform, which has been designed for protective nucleic acid encapsulation, augmented biodistribution and PK, and suitable for intravenous (IV) administration. A key design was to introduce cationic CD rings onto a multi-arm PEG backbone in a spatially selective fashion. The optimal structural design was obtained through iterative rounds of experimentation to determine the appropriate type and density of cationic charge on CD ring, the degree of PEGylation, the size and structure of polymer backbone, etc. This allowed us to effectively deliver large size reporter and therapeutic plasmids in cancer mouse models. Post IV injection, we demonstrated that our multi-arm polymer design significantly enhanced circulatory half-life and PK profile compared to the linear PRX. We continued to use the multi-arm PRX to formulate a therapeutic plasmid encoding an immunomodulatory cytokine, IL-12. When tested in a colon cancer syngeneic mouse model with same background, the IL-12 plasmid was protected by the multi-arm PRX and delivered through the tail vein to the tumor site, leading to a significant tumor inhibition effect. Moreover, our delivery system was devoid of major systemic toxicity.

Irinotecan is a key chemotherapeutic agent for the treatment of colorectal (CRC) and pancreatic (PDAC) cancer. Because of a high incidence of bone marrow and gastrointestinal (GI) toxicity, Onivyde (a liposome) was introduced to provide encapsulated irinotecan (Ir) delivery in PDAC patients. While there is an ongoing clinical trial (NCT02551991) to investigate the use of Onivyde as a first-line option to replace irinotecan in FOLFIRINOX, the liposomal formulation is currently prescribed as a second-line treatment option (in combination with 5-fluorouracil and leucovorin) for patients with metastatic PDAC who failed gemcitabine therapy. However, the toxicity of Onivyde remains a concern that needs to be addressed for use in CRC as well. Our goal was to custom design a mesoporous silica nanoparticle (MSNP) carrier for encapsulated irinotecan delivery in a robust CRC model. This was achieved by developing an orthotopic tumor chunk model in immunocompetent mice. With a view to increase the production volume and to expand the disease applications, the carrier design was improved by using an ethanol exchange method for coating of a supported lipid bilayer (LB) that entraps a protonating agent. The encapsulated protonating agent was subsequently used for remote loading of irinotecan. The excellent irinotecan loading capacity and stability of the LB-coated MSNP carrier, also known as a "silicasome", previously showed improved efficacy and reduced toxicity when compared to an in-house liposomal carrier in a PDAC model. Intravenous injection of the silicasomes in a well-developed orthotopic colon cancer model in mice demonstrated improved pharmacokinetics and tumor drug content over free drug and Onivyde. Moreover, improved drug delivery was accompanied by substantially improved efficacy, increased survival, and reduced bone marrow and GI toxicity compared to the free drug and Onivyde. We also confirmed that the custom-designed irinotecan silicasomes outperform Onivyde in an orthotopic PDAC model. In summary, the Ir-silicasome appears to be promising as a treatment option for CRC in humans based on improved efficacy and the carrier's favorable safety profile.

Nanoparticles (NPs) can be used to accomplish antigen-specific immune tolerance in allergic and autoimmune disease. The available options for custom-designing tolerogenic NPs include the use of nanocarriers that introduce antigens into natural tolerogenic environments, such as the liver, where antigen presentation promotes tolerance to self- or foreign antigens. Here, we demonstrate the engineering of a biodegradable polymeric poly(lactic- co-glycolic acid) (PLGA) nanocarrier for the selective delivery of the murine allergen, ovalbumin (OVA), to the liver. This was accomplished by developing a series of NPs in the 200-300 nm size range as well as decorating particle surfaces with ligands that target scavenger and mannose receptors on liver sinusoidal endothelial cells (LSECs). LSECs represent a major antigen-presenting cell type in the liver capable of generating regulatory T-cells (Tregs).  In vitro exposure of LSECs to NP^OVA induced abundant TGF-β, IL-4, and IL-10 production, which was further increased by surface ligands. Animal experiments showed that, in the chosen size range, NP^OVA was almost exclusively delivered to the liver, where the colocalization of fluorescent-labeled particles with LSECs could be seen to increase by surface ligand decoration. Moreover, prophylactic treatment with NP^OVA in OVA-sensitized and challenged animals (aerosolized inhalation) could be seen to significantly suppress anti-OVA IgE responses, airway eosinophilia, and TH2 cytokine production in the bronchoalveolar lavage fluid. The suppression of allergic airway inflammation was further enhanced by attachment of surface ligands, particularly for particles decorated with the ApoB peptide, which induced high levels of TGF-β production in the lung along with the appearance of Foxp3⁺ Tregs. The ApoB-peptide-coated NPs could also interfere in allergic airway inflammation when delivered postsensitization. The significance of these findings is that liver and LSEC targeting PLGA NPs could be used for therapy of allergic airway disease, in addition to the potential of using their tolerogenic effects for other disease applications.

Carbon nanotubes (CNTs) exhibit a number of physicochemical properties that contribute to adverse biological outcomes. However, it is difficult to define the independent contribution of individual properties without purified materials. A library of highly purified single-walled carbon nanotubes (SWCNTs) of different lengths is prepared from the same base material by density gradient ultracentrifugation, designated as short (318 nm), medium (789 nm), and long (1215 nm) SWCNTs. In vitro screening shows length-dependent interleukin-1β (IL-1β) production, in order of long > medium > short. However, there are no differences in transforming growth factor-β1 production in BEAS-2B cells. Oropharyngeal aspiration shows that all the SWCNTs induce profibrogenic effects in mouse lung at 21 d postexposure, but there are no differences between tube lengths. In contrast, these SWCNTs demonstrate length-dependent antibacterial effects on Escherichia coli, with the long SWCNT exerting stronger effects than the medium or short tubes. These effects are reduced by Pluronic F108 coating or supplementing with glucose. The data show length-dependent effects on proinflammatory response in macrophage cell line and antibacterial effects, but not on collagen deposition in the lung. These data demonstrate that over the length scale tested, the biological response to highly purified SWCNTs is dependent on the complexity of the nano/bio interface.

We have recently shown that the toxicological potential of GaAs and InAs particulates in cells is size- and dissolution-dependent, tending to be more pronounced for nano- vs micron-sized particles. Whether the size-dependent dissolution and shedding of ionic III-V materials also apply to pulmonary exposure is unclear. While it has been demonstrated that micron-sized III-V particles, such as GaAs and InAs, are capable of inducing hazardous pulmonary effects in an occupational setting as well as in animal studies, the effect of submicron particles (e.g., the removal of asperities during processing of semiconductor wafers) is unclear. We used cytokine profiling to compare the pro-inflammatory effects of micron- and nanoscale GaAs and InAs particulates in cells as well as the murine lung 40 h and 21 days after oropharyngeal aspiration. Use of cytokine array technology in macrophage and epithelial cell cultures demonstrated a proportionally higher increase in the levels of matrix metalloproteinase inducer (EMMPRIN), macrophage migration inhibitory factor (MIF), and interleukin 1β (IL-1β) by nanosized (n) GaAs and n-InAs as well as As(III). n-GaAs and n-InAs also triggered higher neutrophil counts in the bronchoalveolar lavage fluid (BALF) of mice than micronscale particles 40 h post-aspiration, along with increased production of EMMPRIN and MIF. In contrast, in animals sacrificed 21 days after exposure, only n-InAs induced fibrotic lung changes as determined by increased lung collagen as well as increased levels of TGF-β1 and PDGF-AA in the BALF. A similar trend was seen for EMMPRIN and matrix metallopeptidase (MMP-9) levels in the BALF. Nano- and micron-GaAs had negligible subacute effects. Importantly, the difference between the 40 h and 21 days data appears to be biopersistence of n-InAs, as demonstrated by ICP-OES analysis of lung tissue. Interestingly, an ionic form of In, InCl₃, also showed pro-fibrogenic effects due to the formation of insoluble In(OH)₃ nanostructures. All considered, these data indicate that while nanoscale particles exhibit increased pro-inflammatory effects in the lung, most effects are transient, except for n-InAs and insoluble InCl₃ species that are biopersistent and trigger pro-fibrotic effects. These results are of potential importance for the understanding the occupational health effects of III-V particulates.