Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations.

BACKGROUND: Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs. METHODS: 13 hMSC samples derived from 10 "healthy" donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis. RESULTS: scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion. CONCLUSIONS: This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

3D in vitro microvascular model-based lymphoma model.

Diffuse large B-cell lymphoma (DLBCL) is a particularly aggressive cancer, impacting the lives of approximately 20,000 people annually in the United States. Elucidating cellular interactions that occur within the microenvironment of DLBCL tumors is crucial to the successful development of therapeutic strategies for this condition. As the in vivo microenvironment of DLBCL is quite complex and variable, in vitro platforms that can sufficiently recapitulate these multifaceted cellular interactions without introducing the complexities of in vivo systems are vital for understanding the pathophysiology of this disease. In this chapter, we present a method for fabrication and development of an in vitro DLBCL-on-chip model in which a fully vascularized, perfusable, microfluidic traverses a DLBCL tumor cell-laden hydrogel that successfully recapitulates hallmark attributes and cellular interaction that occur within the DLBCL tumor microenvironment. As this microfluidic approach makes use of common laboratory items and does not require traditional photolithography to fabricate, this system represents a vital tool that can unlock previously inaccessible research areas of the DLBCL tumor microenvironment to researchers across numerous fields.

A synthetic stroma-free germinal center niche for efficient generation of humoral immunity ex vivo.

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial "follicular T helper cells (TFH)" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40 and by mimicking artificial "follicular dendritic cells (FDC)" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

A simple, clinically relevant therapeutic vaccine shows long-term protection in an aggressive, delayed-treatment B lymphoma model.

Despite initial remission after successful treatments, B lymphoma patients often encounter relapses and resistance causing high mortality. Thus, there is a need to develop therapies that prevent relapse by providing long-term protection and, ultimately, lead to functional cure. In this study, our goal was to develop a simple, clinically relevant, and easily translatable therapeutic vaccine that provides durable immune protection against aggressive B cell lymphoma and identify critical immune biomarkers that are predictive of long-term survival. In a delayed-treatment, aggressive, murine model of A20 B lymphoma that mimics human diffuse large B cell lymphoma, we show that therapeutic A20 lysate vaccine adjuvanted with an NKT cell agonist, α-galactosylceramide (α-GalCer), provides long-term immune protection against lethal tumor challenges and the antitumor immunity is primarily CD8 T cell dependent. Using experimental and computational methods, we demonstrate that the initial strength of germinal center reaction and the magnitude of class-switching into a Th1 type humoral response are the best predictors for the long-term immunity of B lymphoma lysate vaccine. Our results not only provide fundamentally insights for successful immunotherapy and long-term protection against B lymphomas, but also present a simple, therapeutic vaccine that can be translated easily due to the facile and inexpensive method of preparation.

Reconstitution of Low-Density Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs into Cancer Cells.

Low-density lipoproteins (LDLs) are a class of nanocarriers for the targeted delivery of therapeutics into aberrant cells that overexpress the LDL receptor. A facile procedure is used for reconstituting the hydrophobic core of LDLs with a binary fatty acid mixture. Facilitated by the tumor targeting capability of the apolipoprotein, the reconstituted, drug-loaded LDLs can effectively target cancer cells that overexpress the LDL receptor while showing minor adverse impact on normal fibroblasts. According to a hypothesized mechanism, the reconstituted LDLs can also enable metabolism-triggered drug release while preventing the payloads from lysosomal degradation. This study demonstrates that LDLs reconstructed with fatty acids hold great promise to serve as effective and versatile nanocarriers for targeted cancer therapy.

3D microvascular model recapitulates the diffuse large B-cell lymphoma tumor microenvironment in vitro.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer that affects ∼22 000 people in the United States yearly. Understanding the complex cellular interactions of the tumor microenvironment is critical to the success and development of DLBCL treatment strategies. In vitro platforms that successfully model the complex tumor microenvironment without introducing the variability of in vivo systems are vital for understanding these interactions. To date, no such in vitro model exists that can accurately recapitulate the interactions that occur between immune cells, cancer cells, and endothelial cells in the tumor microenvironment of DLBCL. To that end, we developed a lymphoma-on-chip model consisting of a hydrogel based tumor model traversed by a vascularized, perfusable, round microchannel that successfully recapitulates key complexities and interactions of the in vivo tumor microenvironment in vitro. We have shown that the perfusion capabilities of this technique allow us to study targeted treatment strategies, as well as to model the diffusion of infused reagents spatiotemporally. Furthermore, this model employs a novel fabrication technique that utilizes common laboratory materials, and allows for the microfabrication of multiplex microvascular environments without the need for advanced microfabrication facilities. Through our facile microfabrication process, we are able to achieve micro vessels within a tumor model that are highly reliable and precise over the length of the vessel. Overall, we have developed a tool that enables researchers from many diverse disciplines to study previously inaccessible aspects of the DLBCL tumor microenvironment, with profound implications for drug delivery and design.

Surface-Presentation of CpG and Protein-Antigen on Pathogen-Like Polymer Particles Generate Strong Prophylactic and Therapeutic Antitumor Protection.

Despite significant efforts, development of clinically relevant prophylactic and therapeutic cancer vaccines has proven challenging. Cancer-associated antigens, which are often self-antigens, do not activate innate immune cells sufficiently, underscoring the need for codelivery of appropriate immune-stimulatory adjuvants. Recent research has underscored the need for biomaterial-based carriers for vaccine delivery, not only to target antigens and adjuvants to antigen-presenting cells or to create "depot" like systems but also to avoid acute systemic toxicity of molecular adjuvants that occurs when adjuvants are delivered in their "naked" form. The work presented here focuses on surface-presentation of both antigens and adjuvants on a pathogen-like particle (PLP) platform and understanding how PLP-induced antitumor responses differ when protein antigens and adjuvants, specifically the TLR9 agonist CpG, are delivered on the surface of the same particle (dual-loaded) versus being codelivered on separate particles. Surface-presentation allows easier access of antigens and adjuvants to intracellular targets (e.g., to TLR9 in the phagosomal compartments) and also allows controlled multivalent presentation. Our results show that, surface presentation, as opposed to soluble molecules, was more efficient in activating dendritic cells (DCs) and polarizing them toward generating a stronger cytotoxic T cell response. Signaling and DC polarization between separate and dual-loaded particles were similar, although NF-kB signaling at higher doses was stronger in dual-loaded PLPs. In vivo, dual loaded PLPs performed better than separately loaded PLPs in a prophylactic tumor model of melanoma and were comparable to immunization using incomplete Freud's adjuvant (IFA). In contrast both PLP-based delivery modalities performed similarly in a therapeutic melanoma-vaccine model and significantly outperformed IFA-based vaccination. These results indicate that surface-presentation of antigens and adjuvants on polymer-particles is a promising modality for efficient anticancer vaccines.

Targeted Magnetic Liposomes Loaded with Doxorubicin.

Targeted delivery systems for anticancer drugs are urgently needed to achieve maximum therapeutic efficacy by site-specific accumulation and thereby minimizing adverse effects resulting from systemic distribution of many potent anticancer drugs. We have prepared folate receptor-targeted magnetic liposomes loaded with doxorubicin, which are designed for tumor targeting through a combination of magnetic and biological targeting. Furthermore, these liposomes are designed for hyperthermia-induced drug release to be mediated by an alternating magnetic field and to be traceable by magnetic resonance imaging (MRI). Here, detailed preparation and relevant characterization techniques of targeted magnetic liposomes encapsulating doxorubicin are described.

The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma.

Success of an immunotherapy for cancer often depends on the critical balance of T helper 1 (Th1) and T helper 2 (Th2) responses driven by antigen presenting cells, specifically dendritic cells (DCs). Th1-driven cytotoxic T cell (CTL) responses are key to eliminating tumor cells. It is well established that CpG oligonucleotides (ODN), a widely studied Toll-like receptor 9 (TLR9) agonist, used to enhance Th1 response, also induces high levels of the anti-inflammatory, Th2-promoting cytokine IL10, which could dampen the resulting Th1 response. Biomaterials-based immunomodulatory strategies that can reduce IL10 production while maintaining IL12 levels during CpG delivery could further enhance the Th1/Th2 cytokine balance and improve anti-tumor immune response. Here we report that dual-delivery of IL10-silencing siRNA along with CpG ODN to the same DCs using pathogen-mimicking microparticles (PMPs), significantly enhances their Th1/Th2 cytokine ratio through concurrent inhibition of CpG-induced IL10 production. Co-delivery of poly(I:C), a TLR3 agonist had only minor effects on IL10 levels. Further, simultaneous immunotherapy with CpG ODN and IL10 siRNA enhanced immune protection of an idiotype DNA vaccine in a prophylactic murine model of B cell lymphoma whereas co-delivery of poly(I:C) and CpG did not enhance protection. These results suggest that PMPs can be used to precisely modulate TLR ligand-mediated immune-stimulation in DCs, through co-delivery of cytokine-silencing siRNAs and thereby boost antitumor immunity.

Synthesis of hydrophilic superparamagnetic magnetite nanoparticles via thermal decomposition of Fe(acac), in 80 vol% TREG + 20 vol% TREM.

In this paper, we report single step synthesis of hydrophilic superparamagnetic magnetite nanoparticles by thermolysis of Fe(acac)3 and their characterization of the properties relevant to biomedical applications like hyperthermia and magnetic resonance imaging (MRI). Size and morphology of the particles were determined by Transmission electron microscopy (TEM) while phase purity and structure of the particles were identified by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Magnetic properties were evaluated using vibrating sample magnetometer (VSM) and superconducting quantum interference device (SQUID) measurements. The as prepared nanoparticles were found to be superparamagnetic with the blocking temperature of 136 K and were easily suspendable in water. Cytotoxicity studies on human cervical (SiHa), mouse melanoma (B16F10) and mouse primary fibroblast cells demonstrated that up to a dose of 0.1 mg/ml, the magnetite nanoparticles were nontoxic to the cells. To evaluate the feasibility of their uses in hyperthermia and MRI applications, specific absorption rate (SAR) and spin-spin relaxation time (T2) were measured respectively. SAR has been calculated to be above 80 Watt/g for samples with the iron concentration of 5-20 mg/ml at 10 kA/m AC magnetic field and 425 kHz frequency. r2 relaxivity value was measured as 358.4 mM(-1)S(-1) which is almost double as compared to that of the Resovist, a commercially available MRI contrast agent. Thus the as-prepared magnetite nanoparticles may be used for hyperthermia and MRI applications due to their promising SAR and r2 values.

Thermal behavior of magnetically modalized poly(N-isopropylacrylamide)-chitosan based nanohydrogel.

Poly(NIPAAm)-CS based nanohydrogels (NHGs) and iron oxide (Fe(3)O(4)) magnetic nanoparticles encapsulated magnetic nanohydrogels (MNHGs) were synthesized by free radical polymerization of N-isopropylacrylamide (NIPAAm) at 60 degrees C in presence of chitosan (CS) in different feed ratios. The polymerization of NIPAAm and the presence of CS as well as Fe(3)O(4) in hydrogels were confirmed from Fourier transform infrared (FTIR) spectra and X-ray diffraction (XRD), respectively. (13)C solid state nuclear magnetic resonance (NMR) spectra clearly revealed the grafting of CS into poly(NIPAAm). The scanning electron microscopy (SEM) and atomic force microscopy (AFM) images showed the formation of spherical shaped NHGs of different sizes ranging from 50 nm to 200 nm depending upon the feed ratios of CS and NIPAAm, which was further supported by mean hydrodynamic diameter measured by dynamic light scattering (DLS). It has been observed that CS not only served as a cross linker during polymerization but also plays a critical role in controlling the growth of NHG and enhancement in lower critical solution temperature (LCST). The encapsulation of Fe(3)O(4) nanoparticles (10-12 nm) into NHGs ( approximately 200 nm) was confirmed by transmission electron microscopy (TEM) and further corroborated with magnetic force microscopy (MFM) image. The LCST of poly(NIPAAm) was found to increase with increasing weight ratio of CS to NIPAAm. Furthermore, the encapsulation of iron oxide nanoparticles into hydrogels also caused an increment in LCST. Specifically, temperature optimized NHG and MNHG were fabricated having LCST close to 42 degrees C (hyperthermia temperature). The MNHG shows optimal magnetization, good specific absorption rate (under external AC magnetic field) and excellent cytocompatibility with L929 cell lines, which may find potential applications in hyperthermia treatment of cancer and targeted drug delivery.

Targeted magnetic liposomes loaded with doxorubicin.

Targeted delivery systems for anticancer drugs are urgently needed to achieve maximum therapeutic efficacy by site-specific accumulation and thereby minimizing adverse effects resulting from systemic distribution of many potent anticancer drugs. We have prepared folate receptor targeted magnetic liposomes loaded with doxorubicin, which are designed for tumor targeting through a combination of magnetic and biological targeting. Furthermore, these liposomes are designed for hyperthermia-induced drug release to be mediated by an alternating magnetic field and to be traceable by magnetic resonance imaging (MRI). Here, detailed preparation and relevant characterization techniques of targeted magnetic liposomes encapsulating doxorubicin are described.

Targeted temperature sensitive magnetic liposomes for thermo-chemotherapy.

We describe folate receptor targeted thermosensitive magnetic liposomes, which are designed to combine features of biological and physical (magnetic) drug targeting for use in magnetic hyperthermia-triggered drug release. The optimized liposome formulation DPPC:cholesterol:DSPE-PEG(2000):DSPE-PEG(2000)-Folate at 80:20:4.5:0.5 molar ratio showed calcein release of about 70% both in PBS and in 50% FBS (fetal bovine serum) at 43 degrees C and less than 5% release at 37 degrees C following 1h incubation. Folate-targeted doxorubicin-containing magnetic liposomes of the above lipid composition (MagFolDox) showed encapsulation efficiencies of about 85% and 24% for doxorubicin and magnetic nanoparticles (mean crystallite size 10nm), respectively. This magnetic formulation displayed the desired temperature sensitivity with 52% doxorubicin release in 50% fetal bovine serum (FBS) following 1h incubation at 43 degrees C. MagFolDox, when physically targeted to tumor cells in culture by a permanent magnetic field yielded a substantial increase in cellular uptake of doxorubicin as compared to Caelyx (a commercially available liposomal doxorubicin preparation), non-magnetic folate-targeted liposomes (FolDox) and free doxorubicin in folate receptor expressing tumor cell lines (KB and HeLa cells). This resulted in a parallel increase in cytotoxicity over Caelyx and FolDox. Magnetic hyperthermia at 42.5 degrees C and 43.5 degrees C synergistically increased the cytotoxicity of MagFolDox. The results suggest that an integrated concept of biological and physical drug targeting, triggered drug release and hyperthermia based on magnetic field influence can be used advantageously for thermo-chemotherapy of cancers.

Comparative evaluation of heating ability and biocompatibility of different ferrite-based magnetic fluids for hyperthermia application.

In this study, lauric acid-coated, superparamagnetic, nanoparticle-based magnetic fluids of different ferrites (Fe(3)O(4), MnFe(2)O(4), and CoFe(2)O(4)) were prepared and compared in terms of heating ability and biocompatibility to evaluate the feasibility of use in hyperthermia treatment of cancer. All the magnetic fluids prepared had particles of average sizes 9-11 nm. Heating ability of these magnetic fluids was evaluated by calorimetric measurement of specific absorption rate (SAR) at 300 kHz frequency and 15 kA/m field. Fe(3)O(4) and MnFe(2)O(4) showed higher SAR (120 and 97 W/g of ferrite, respectively) than CoFe(2)O(4) (37 W/g of ferrite). In vitro study on BHK 21 cell lines showed dose-dependent cell viability for all the magnetic fluids. Threshold-biocompatible ferrite concentration for all the magnetic fluids was 0.1 mg/mL. Above 0.2 mg/mL, CoFe(2)O(4) was more toxic than the other magnetic fluids. On intravenous injection of different doses (50, 200, and 400 mg/kg body weight) of magnetic fluids in mice, no significant changes in hematological and biochemical parameters were observed for Fe(3)O(4) and MnFe(2)O(4). With CoFe(2)O(4), an increase in SGPT levels at a dose rate of 400 mg/kg body weight was observed, indicating its mild hepatotoxic effect. However, histology of different vital organs showed no pathological changes for all the three magnetic fluids. Further, long term in vivo evaluation of biocompatibility of the lauric acid-coated ferrites is warranted. This study shows that lauric acid-coated, superparamagnetic Fe(3)O(4) and MnFe(2)O(4) may be used for hyperthermia treatment and are to be preferred over CoFe(2)O(4).