In vivo bone marrow microenvironment siRNA delivery using lipid-polymer nanoparticles for multiple myeloma therapy.

Multiple myeloma (MM), a hematologic malignancy that preferentially colonizes the bone marrow, remains incurable with a survival rate of 3 to 6 mo for those with advanced disease despite great efforts to develop effective therapies. Thus, there is an urgent clinical need for innovative and more effective MM therapeutics. Insights suggest that endothelial cells within the bone marrow microenvironment play a critical role. Specifically, cyclophilin A (CyPA), a homing factor secreted by bone marrow endothelial cells (BMECs), is critical to MM homing, progression, survival, and chemotherapeutic resistance. Thus, inhibition of CyPA provides a potential strategy to simultaneously inhibit MM progression and sensitize MM to chemotherapeutics, improving therapeutic response. However, inhibiting factors from the bone marrow endothelium remains challenging due to delivery barriers. Here, we utilize both RNA interference (RNAi) and lipid-polymer nanoparticles to engineer a potential MM therapy, which targets CyPA within blood vessels of the bone marrow. We used combinatorial chemistry and high-throughput in vivo screening methods to engineer a nanoparticle platform for small interfering RNA (siRNA) delivery to bone marrow endothelium. We demonstrate that our strategy inhibits CyPA in BMECs, preventing MM cell extravasation in vitro. Finally, we show that siRNA-based silencing of CyPA in a murine xenograft model of MM, either alone or in combination with the Food and Drug Administration (FDA)-approved MM therapeutic bortezomib, reduces tumor burden and extends survival. This nanoparticle platform may provide a broadly enabling technology to deliver nucleic acid therapeutics to other malignancies that home to bone marrow.

Naked mole-rats lack cold sensitivity before and after nerve injury.

Neuropathic pain is a chronic disease state resulting from injury to the nervous system. This type of pain often responds poorly to standard treatments and occasionally may get worse instead of better over time. Patients who experience neuropathic pain report sensitivity to cold and mechanical stimuli. Since the nociceptive system of African naked mole-rats contains unique adaptations that result in insensitivity to some pain types, we investigated whether naked mole-rats may be resilient to sensitivity following nerve injury. Using the spared nerve injury model of neuropathic pain, we showed that sensitivity to mechanical stimuli developed similarly in mice and naked mole-rats. However, naked mole-rats lacked sensitivity to mild cold stimulation after nerve injury, while mice developed robust cold sensitivity. We pursued this response deficit by testing behavior to activators of transient receptor potential (TRP) receptors involved in detecting cold in naïve animals. Following mustard oil, a TRPA1 activator, naked mole-rats responded similarly to mice. Conversely, icilin, a TRPM8 agonist, did not evoke pain behavior in naked mole-rats when compared with mice. Finally, we used RNAscope to probe for TRPA1 and TRPM8 messenger RNA expression in dorsal root ganglia of both species. We found increased TRPA1 messenger RNA, but decreased TRPM8 punctae in naked mole-rats when compared with mice. Our findings likely reflect species differences due to evolutionary environmental responses that are not easily explained by differences in receptor expression between the species.

Evaluating the Mechanisms of Light-Triggered siRNA Release from Nanoshells for Temporal Control Over Gene Regulation.

The ability to regulate intracellular gene expression with exogenous nucleic acids such as small interfering RNAs (siRNAs) has substantial potential to improve the study and treatment of disease. However, most transfection agents and nanoparticle-based carriers that are used for the intracellular delivery of nucleic acids cannot distinguish between diseased and healthy cells, which may cause them to yield unintended widespread gene regulation. An ideal delivery system would only silence targeted proteins in diseased tissue in response to an external stimulus. To enable spatiotemporal control over gene silencing, researchers have begun to develop nucleic acid-nanoparticle conjugates that keep their nucleic acid cargo inactive until it is released from the nanoparticle on-demand by externally applied near-infrared laser light. This strategy can overcome several limitations of other nucleic acid delivery systems, but the mechanisms by which these platforms operate remain ill understood. Here, we perform a detailed investigation of the mechanisms by which silica core/gold shell nanoshells (NSs) release conjugated siRNA upon excitation with either pulsed or continuous wave (CW) near-infrared (NIR) light, with the goal of providing insight into how these nanoconjugates can enable on-demand gene regulation. We demonstrate that siRNA release from NSs upon pulsed laser irradiation is a temperature-independent process that is substantially more efficient than siRNA release triggered by CW irradiation. Contrary to literature, which suggests that only pulsed irradiation releases siRNA duplexes, we found that both modes of irradiation release a mixture of siRNA duplexes and single-stranded oligonucleotides, but that pulsed irradiation results in a higher percentage of released duplexes. To demonstrate that the siRNA released from NSs upon pulsed irradiation remains functional, we evaluated the use of NSs coated with green fluorescent protein (GFP)-targeted siRNA (siGFP-NS) for on-demand knockdown of GFP in cells. We found that GFP-expressing cells treated with siGFP-NS and irradiated with a pulsed laser experienced a 33% decrease in GFP expression compared to cells treated with no laser. Further, we observed that light-triggered gene silencing mediated by siGFP-NS is more potent than using commercial transfection agents to deliver siRNA into cells. This work provides unprecedented insight into the mechanisms by which plasmonic NSs release siRNA upon light irradiation and demonstrates the importance of thoroughly characterizing photoresponsive nanosystems for applications in triggered gene regulation.

Photochemotherapeutic Properties of a Linear Tetrapyrrole Palladium(II) Complex displaying an Exceptionally High Phototoxicity Index.

Photodynamic therapy (PDT) represents a minimally invasive and highly localized treatment strategy to ablate tumors with few side effects. In PDT, photosensitizers embedded within tumors are activated by light and undergo intersystem crossing, followed by energy transfer to molecular oxygen, resulting in the production of toxic singlet oxygen (1O2). Previously, we reported a robust, linear tetrapyrrole palladium(II) complex, Pd[DMBil1], characterized by its facile and modular synthesis, broad absorption profile, and efficient 1O2 quantum yield of ΦΔ = 0.8 in organic media. However, the insolubility of this porphyrinoid derivative in aqueous solution prevents its use under biologically relevant conditions. In this work, we report the synthesis of Pd[DMBil1]-PEG750, a water-soluble dimethylbiladiene derivative that is appended with a poly(ethylene) glycol (PEG) functionality. Characterization of this complex shows that this PEGylated biladiene architecture maintains the attractive photophysical properties of the parent complex under biologically relevant conditions. More specifically, the absorption profile of Pd[DMBil1]-PEG750 closely matches that of Pd[DMBil1] and obeys the Beer-Lambert Law, suggesting that the complex does not aggregate under biologically relevant conditions. Additionally, the emission spectrum of Pd[DMBil1]-PEG750 retains the fluorescence and phosphorescence features characteristic of Pd[DMBil1]. Importantly, the PEGylated photosensitizer generates 1O2 with ΦΔ = 0.57, which is well within the range to warrant interrogation as a potential PDT agent for treatment of cancer cells. The Pd[DMBil1]-PEG750 is biologically compatible, as it is taken up by MDA-MB-231 triple negative breast cancer (TNBC) cells and has an effective dose (ED50) of only 0.354 µM when exposed to λex > 500 nm light for 30 min. Impressively, the lethal dose (LD50) of Pd[DMBil1]-PEG750 without light exposure was measured to be 1.87 mM, leading to a remarkably high phototoxicity index of ∼5300, which is vastly superior to existing photosensitizers that form the basis for clinical PDT treatments. Finally, through flow cytometry experiments, we show that PDT with Pd[DMBil1]-PEG750 induces primarily apoptotic cell death in MDA-MB-231 cells. Overall these results demonstrate that Pd[DMBil1]-PEG750 is an easily prepared, biologically compatible, and well-tolerated photochemotherapeutic agent that can efficiently drive the photoinduced apoptotic death of TNBC cells.

Frizzled7 Antibody-Functionalized Nanoshells Enable Multivalent Binding for Wnt Signaling Inhibition in Triple Negative Breast Cancer Cells.

Antibodies that antagonize cell signaling pathways specific to their targeted receptor are invaluable tools to study and treat malignancies, but their utility is limited by high production costs and treatment dosages. Researchers have shown that antibodies conjugated to nanoparticles display increased affinity for their target relative to freely delivered antibodies due to multivalency, and this study investigates how this multivalency can enable antibody-nanoparticle conjugates to inhibit oncogenic cell signaling more effectively than freely delivered antibodies. This effect was evaluated using triple negative breast cancer (TNBC) cells that are characterized by hyperactive Wnt signaling mediated through overexpressed Frizzled7 (FZD7) transmembrane receptors. Through analysis of the expression of ß-catenin and Axin2, two downstream targets in the Wnt pathway, the results demonstrate that FZD7 antibody-nanoshell conjugates (FZD7-NS) are drastically more effective at inhibiting Wnt signaling in TNBC cells than freely delivered FZD7 antibodies. Additionally, cells treated with FZD7-NS, but not cells treated with freely delivered FZD7 antibodies, have decreased viability, indicating the therapeutic potential of this technology. The results demonstrate that antibody-functionalized nanoparticles can exploit multivalency for improved signal cascade interference over free antibodies, and this may ultimately permit lower antibody dosages to be administered to study signaling pathways or to manage diseases.

Systematic development of ionizable lipid nanoparticles for placental mRNA delivery using a design of experiments approach.

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs are only in the beginning stages of development for safe and effective mRNA delivery to the placenta to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that utilizing C12-200 as the ionizable lipid and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the phospholipid in the LNP design yields high transfection efficiency in vitro. Analysis of lipid molar composition as a design parameter in LNPs displayed a strong correlation between apparent pKa and poly (ethylene) glycol (PEG) content, as a reduction in PEG molar amount increases apparent pKa. Further, we present one LNP platform that exhibits the highest delivery of placental growth factor mRNA to the placenta in pregnant mice, resulting in synthesis and secretion of a potentially therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta, and our top LNP formulations may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

Antibody and siRNA Nanocarriers to Suppress Wnt Signaling, Tumor Growth, and Lung Metastasis in Triple-Negative Breast Cancer.

The paucity of targeted therapies for triple-negative breast cancer (TNBC) causes patients with this aggressive disease to suffer a poor clinical prognosis. A promising target for therapeutic intervention is the Wnt signaling pathway, which is activated in TNBC cells when extracellular Wnt ligands bind overexpressed Frizzled7 (FZD7) transmembrane receptors. This stabilizes intracellular ß-catenin proteins that in turn promote transcription of oncogenes that drive tumor growth and metastasis. To suppress Wnt signaling in TNBC cells, we developed therapeutic nanoparticles (NPs) functionalized with FZD7 antibodies and ß-catenin small interfering RNAs (siRNAs). The antibodies enable TNBC cell-specific binding and inhibit Wnt signaling by locking FZD7 receptors in a ligand unresponsive state, while the siRNAs suppress ß-catenin through RNA interference. Compared to NPs coated with antibodies or siRNAs individually, NPs coated with both agents more potently reduce the expression of several Wnt related genes in TNBC cells, leading to greater inhibition of cell proliferation, migration, and spheroid formation. In two murine models of metastatic TNBC, the dual antibody/siRNA nanocarriers outperformed controls in terms of inhibiting tumor growth, metastasis, and recurrence. These findings demonstrate suppressing Wnt signaling at both the receptor and mRNA levels via antibody/siRNA nanocarriers is a promising approach to combat TNBC.

siRNA Lipid-Polymer Nanoparticles Targeting E-Selectin and Cyclophilin A in Bone Marrow for Combination Multiple Myeloma Therapy.

Introduction: Multiple myeloma (MM) is a hematological blood cancer of the bone marrow that remains largely incurable, in part due to its physical interactions with the bone marrow microenvironment. Such interactions enhance the homing, proliferation, and drug resistance of MM cells. Specifically, adhesion receptors and homing factors, E-selectin (ES) and cyclophilin A (CyPA), respectively, expressed by bone marrow endothelial cells enhance MM colonization and dissemination. Thus, silencing of ES and CyPA presents a potential therapeutic strategy to evade MM spreading. However, small molecule inhibition of ES and CyPA expressed by bone marrow endothelial cells remains challenging, and blocking antibodies induce further MM propagation. Therefore, ES and CyPA are promising candidates for inhibition via RNA interference (RNAi). Methods: Here, we utilized a previously developed lipid-polymer nanoparticle for RNAi therapy, that delivers siRNA to the bone marrow perivascular niche. We utilized our platform to co-deliver ES and CyPA siRNAs to prevent MM dissemination in vivo. Results: Lipid-polymer nanoparticles effectively downregulated ES expression in vitro, which decreased MM cell adhesion and migration through endothelial monolayers. Additionally, in vivo delivery of lipid-polymer nanoparticles co-encapsulating ES and CyPA siRNA extended survival in a xenograft mouse model of MM, either alone or in combination with the proteasome inhibitor bortezomib. Conclusions: Our combination siRNA lipid-polymer nanoparticle therapy presents a vascular microenvironment-targeting strategy as a potential paradigm shift for MM therapies, which could be extended to other cancers that colonize the bone marrow. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00774-y.

Lipid Nanoparticle Composition Drives mRNA Delivery to the Placenta.

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs have yet to be developed for safe and effective mRNA delivery to the placenta as a method to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that a specific combination of ionizable lipid and phospholipid in the LNP design yields high transfection efficiency in vitro . Further, we present one LNP platform that exhibits highest delivery of placental growth factor mRNA to the placenta in pregnant mice, which demonstrates induced protein synthesis and secretion of a therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta. Our top LNPs may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg-1, or intravenously (IV), 2 mg kg-1, and then IV administer modmRNA-LNP, 0.32 mg kg-1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.

Checkpoint blockade-induced CD8+ T cell differentiation in head and neck cancer responders.

BACKGROUND: Immune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%-20% of patients and underpinnings of resistance remain undefined. METHODS: Starting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models. RESULTS: Anti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1- (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions. CONCLUSIONS: These data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

Ionizable lipid nanoparticles for in utero mRNA delivery.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

Yap1 Mediates Trametinib Resistance in Head and Neck Squamous Cell Carcinomas.

PURPOSE: In a head and neck squamous cell carcinoma (HNSCC) "window of opportunity" clinical trial, we reported that trametinib reduced MEK-Erk1/2 activation and resulted in tumor responses in a subset of patients. Here, we investigated resistance to trametinib and molecular correlates in HNSCC cell lines and patient samples. EXPERIMENTAL DESIGN: HNSCC cell lines were treated with trametinib to generate resistant lines. Candidate bypass pathways were assessed using immunoblotting, CRISPR knockout, and survival assays. Effectiveness of combined trametinib and verteporfin targeting was evaluated. Patient-derived xenografts (PDXs) from responder patients were treated with trametinib and resistant tumors were analyzed. Window trial clinical samples were subjected to whole-exome and RNA sequencing. RESULTS: HNSCC cell lines developed resistance (CAL27-TR and HSC3-TR) after prolonged trametinib exposure. Downstream effectors of the Hippo pathway were activated in CAL27-TR and HSC3-TR, and combined trametinib and verteporfin treatment resulted in synergistic treatment response. We defined the Hippo pathway effector Yap1 as an induced survival pathway promoting resistance to trametinib in HSC3-TR. Yap1 was necessary for HSC3-TR trametinib resistance, and constitutively active Yap1 was sufficient to confer resistance in parental HSC3. Analysis of trametinib neoadjuvant trial patient tumors indicated canonical MEK-Erk1/2 pathway activating mutations were infrequent, and Yap1 activity increased following trametinib treatment. Trametinib treatment of a PDX from a responder patient resulted in evolution of resistance with increased Yap1 expression and activity. CONCLUSIONS: These studies identify a Yap1-dependent resistance to trametinib therapy in HNSCCs. Combined Yap1 and MEK targeting may represent a strategy to enhance HNSCC response.

University-community mental health center collaboration: encouraging the dissemination of empirically-based treatment and practice.

Community Mental Health Centers (CMHC) are valuable resources for urban youth and families across the nation. Community demands for high volumes of clinical service, however, often render these agencies without ability to fully evaluate provided services or conduct rigorous research with their target populations. This report asserts the importance of establishing effective collaborations between research-oriented universities and CMHCs in an effort to bridge the gap between empirically-based treatments and "real world" clinical practice. Furthermore, this report explains the establishment of a mutually informative and beneficial university-CMHC collaboration between the Disruptive Behavior Clinic at the University of Illinois-Chicago and the Community Mental Health Council (CMHC) on an evidence-based outpatient family therapy protocol for urban youth with behavior problems. Values and guidelines to facilitate evidence-based practice for fellow academic institutions are asserted. University-CMHC collaboration is an effective way to bring empirically-based practice to the "real world," front-line practice settings of community-based agencies that serve urban youth. Further research with regard to the maintenance and sustainability of these collaborations is suggested.

Targeting EZH2 Enhances Antigen Presentation, Antitumor Immunity, and Circumvents Anti-PD-1 Resistance in Head and Neck Cancer.

PURPOSE: Anti-programmed death-1 (PD-1) receptor-based therapeutics improve survival in patients with recurrent head and neck squamous cell carcinoma (HNSCC), but many do not benefit due to a low response rate. Herein, we identified EZH2 as a therapeutic target that enhanced tumor cell antigen presentation and subsequently sensitized resistant tumors to anti-PD-1 therapy. EXPERIMENTAL DESIGN: EZH2 regulation of antigen presentation was defined using EZH2 inhibitors (GSK126 and EPZ6438) in human and mouse HNSCC cell lines. Mechanistic dissection of EZH2 in regulation of antigen presentation was investigated using flow cytometry, qRT-PCR, ELISA, and chromatin-immunoprecipitation assays. EZH2-deficient cell lines were generated using CRISPR-CAS9. GSK126 and anti-PD-1-blocking antibody were used in testing combinatorial therapy in vivo. RESULTS: EZH2 expression was negatively correlated with antigen-processing machinery pathway components in HNSCC data sets in The Cancer Genome Atlas. EZH2 inhibition resulted in significant upregulation of MHC class I expression in human and mouse human papillomavirus-negative HNSCC lines in vitro and in mouse models in vivo. Enhanced antigen presentation on the tumor cells by EZH2 inhibitors or CRISPR-mediated EZH2 deficiency increased antigen-specific CD8+ T-cell proliferation, IFNγ production, and tumor cell cytotoxicity. Mechanistically, EZH2 inhibition reduced the histone H3K27me3 modification on the ß-2-microglobulin promoter. Finally, in an anti-PD-1-resistant model of HNSCC, tumor growth was suppressed with combination therapy. CONCLUSIONS: Our results demonstrated that targeting EZH2 enhanced antigen presentation and was able to circumvent anti-PD-1 resistance. Thus, combining EZH2 targeting with anti-PD-1 may increase therapeutic susceptibility in HNSCC.

Exploiting the placenta for nanoparticle-mediated drug delivery during pregnancy.

A major challenge to treating diseases during pregnancy is that small molecule therapeutics are transported through the placenta and incur toxicities to the developing fetus. The placenta is responsible for providing nutrients, removing waste, and protecting the fetus from toxic substances. Thus, the placenta acts as a biological barrier between the mother and fetus that can be exploited for drug delivery. Nanoparticle technologies provide the opportunity for safe drug delivery during pregnancy by controlling how therapeutics interact with the placenta. In this Review, we present nanoparticle drug delivery technologies specifically designed to exploit the placenta as a biological barrier to treat maternal, placental, or fetal diseases exclusively, while minimizing off-target toxicities. Further, we discuss opportunities, challenges, and future directions for implementing drug delivery technologies during pregnancy.

Neoadjuvant and Adjuvant Pembrolizumab in Resectable Locally Advanced, Human Papillomavirus-Unrelated Head and Neck Cancer: A Multicenter, Phase II Trial.

PURPOSE: Pembrolizumab improved survival in patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)-unrelated HNSCC. PATIENTS AND METHODS: Neoadjuvant pembrolizumab (200 mg) was administered and followed 2 to 3 weeks later by surgical tumor ablation. Postoperative (chemo)radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (<10%), pTR-1 (10%-49%), and pTR-2 (≥50%). Coprimary endpoints were pTR-2 among all patients and 1-year relapse rate in patients with high-risk pathology (historical: 35%). Correlations of baseline PD-L1 and T-cell infiltration with pTR were assessed. Tumor clonal dynamics were evaluated (ClinicalTrials.gov NCT02296684). RESULTS: Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3-4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among 18 patients with high-risk pathology was 16.7% (95% confidence interval, 3.6%-41.4%). pTR ≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFNγ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0 and confirmed clonal loss in some patients. CONCLUSIONS: Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The 1-year relapse rate in patients with high-risk pathology was lower than historical.

Delivery technologies for cancer immunotherapy.

Immunotherapy has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing, with numerous treatments in clinical and preclinical development. However, a key challenge in the broad implementation of immunotherapies for cancer remains the controlled modulation of the immune system, as these therapeutics have serious adverse effects including autoimmunity and nonspecific inflammation. Understanding how to increase the response rates to various classes of immunotherapy is key to improving efficacy and controlling these adverse effects. Advanced biomaterials and drug delivery systems, such as nanoparticles and the use of T cells to deliver therapies, could effectively harness immunotherapies and improve their potency while reducing toxic side effects. Here, we discuss these research advances, as well as the opportunities and challenges for integrating delivery technologies into cancer immunotherapy, and we critically analyse the outlook for these emerging areas.

Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy.

Immunotherapy has recently emerged as a powerful tool for cancer treatment. Early clinical successes from cancer immunotherapy have led to a growing list of FDA approvals, and many new therapies are in clinical and preclinical development. Nucleic acid therapeutics, including DNA, mRNA, and genome editing systems, hold significant potential as a form of immunotherapy due to its robust use in cancer vaccination, adoptive T-cell therapy, and gene regulation. However, these therapeutics must overcome numerous delivery obstacles to be successful, including rapid in vivo degradation, poor uptake into target cells, required nuclear entry, and potential in vivo toxicity in healthy cells and tissues. Nanoparticle delivery systems have been engineered to overcome several of these barriers as a means to safely and effectively deliver nucleic acid therapeutics to immune cells. In this Review, we discuss the applications of nucleic acid therapeutics in cancer immunotherapy, and we detail how nanoparticle platforms have been designed to deliver mRNA, DNA, and genome editing systems to enhance the potency and safety of these therapeutics.