Enhancing humoral immunity via sustained-release implantable microneedle patch vaccination.

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference.

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

Polyamine-Mediated Stoichiometric Assembly of Ribonucleoproteins for Enhanced mRNA Delivery.

Messenger RNA (mRNA) represents a promising class of nucleic acid drugs. Although numerous carriers have been developed for mRNA delivery, the inefficient mRNA expression inside cells remains a major challenge. Inspired by the dependence of mRNA on 3'-terminal polyadenosine nucleotides (poly A) and poly A binding proteins (PABPs) for optimal expression, we complexed synthetic mRNA containing a poly A tail with PABPs in a stoichiometric manner and stabilized the ribonucleoproteins (RNPs) with a family of polypeptides bearing different arrangements of cationic side groups. We found that the molecular structure of these polypeptides modulates the degree of PABP-mediated enhancement of mRNA expression. This strategy elicits an up to 20-fold increase in mRNA expression in vitro and an approximately fourfold increase in mice. These findings suggest a set of new design principles for gene delivery by the synergistic co-assembly of mRNA with helper proteins.

Engineering a Two-Component Hemostat for the Treatment of Internal Bleeding through Wound-Targeted Crosslinking.

Primary hemostasis (platelet plug formation) and secondary hemostasis (fibrin clot formation) are intertwined processes that occur upon vascular injury. Researchers have sought to target wounds by leveraging cues specific to these processes, such as using peptides that bind activated platelets or fibrin. While these materials have shown success in various injury models, they are commonly designed for the purpose of treating solely primary or secondary hemostasis. In this work, a two-component system consisting of a targeting component (azide/GRGDS PEG-PLGA nanoparticles) and a crosslinking component (multifunctional DBCO) is developed to treat internal bleeding. The system leverages increased injury accumulation to achieve crosslinking above a critical concentration, addressing both primary and secondary hemostasis by amplifying platelet recruitment and mitigating plasminolysis for greater clot stability. Nanoparticle aggregation is measured to validate concentration-dependent crosslinking, while a 1:3 azide/GRGDS ratio is found to increase platelet recruitment, decrease clot degradation in hemodiluted environments, and decrease complement activation. Finally, this approach significantly increases survival relative to the particle-only control in a liver resection model. In light of prior successes with the particle-only system, these results emphasize the potential of this technology in aiding hemostasis and the importance of a holistic approach in engineering new treatments for hemorrhage.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

A new label-free optical imaging method for the lymphatic system enhanced by deep learning.

Our understanding of the lymphatic vascular system lags far behind that of the blood vascular system, limited by available imaging technologies. We present a label-free optical imaging method that visualizes the lymphatic system with high contrast. We developed an orthogonal polarization imaging (OPI) in the shortwave infrared range (SWIR) and imaged both lymph nodes and lymphatic vessels of mice and rats in vivo through intact skin, as well as human mesenteric lymph nodes in colectomy specimens. By integrating SWIR-OPI with U-Net, a deep learning image segmentation algorithm, we automated the lymph node size measurement process. Changes in lymph nodes in response to cancer progression were monitored in two separate mouse cancer models, through which we obtained insights into pre-metastatic niches and correlation between lymph node masses and many important biomarkers. In a human pilot study, we demonstrated the effectiveness of SWIR-OPI to detect human lymph nodes in real time with clinical colectomy specimens. One Sentence Summary: We develop a real-time high contrast optical technique for imaging the lymphatic system, and apply it to anatomical pathology gross examination in a clinical setting, as well as real-time monitoring of tumor microenvironment in animal studies.

Modulating Nanoparticle Size to Understand Factors Affecting Hemostatic Efficacy and Maximize Survival in a Lethal Inferior Vena Cava Injury Model.

Intravenous nanoparticle hemostats offer a potentially attractive approach to promote hemostasis, in particular for inaccessible wounds such as noncompressible torso hemorrhage (NCTH). In this work, particle size was tuned over a range of <100-500 nm, and its effect on nanoparticle-platelet interactions was systematically assessed using in vitro and in vivo experiments. Smaller particles bound a larger percentage of platelets per mass of particle delivered, while larger particles resulted in higher particle accumulation on a surface of platelets and collagen. Intermediate particles led to the greatest platelet content in platelet-nanoparticle aggregates, indicating that they may be able to recruit more platelets to the wound. In biodistribution studies, smaller and intermediate nanoparticles exhibited longer circulation lifetimes, while larger nanoparticles resulted in higher pulmonary accumulation. The particles were then challenged in a 2 h lethal inferior vena cava (IVC) puncture model, where intermediate nanoparticles significantly increased both survival and injury-specific targeting relative to saline and unfunctionalized particle controls. An increase in survival in the second hour was likewise observed in the smaller nanoparticles relative to saline controls, though no significant increase in survival was observed in the larger nanoparticle size. In conjunction with prior in vitro and in vivo experiments, these results suggest that platelet content in aggregates and extended nanoparticle circulation lifetimes are instrumental to enhancing hemostasis. Ultimately, this study elucidates the role of particle size in platelet-particle interactions, which can be a useful tool for engineering the performance of particulate hemostats and improving the design of these materials.

Surface Plasmon Enhanced Upconversion Fluorescence in Short-Wave Infrared for In Vivo Imaging of Ovarian Cancer.

Short-wave infrared (SWIR; 850-1700 nm) upconversion fluorescence enables "autofluorescence-free" imaging with minimal tissue scattering, yet it is rarely explored due to the lack of strongly emissive SWIR upconversion fluorophores. In this work, we apply SWIR upconversion fluorescence for in vivo imaging with exceptional image contrast. Gold nanorods (AuNRs) are used to enhance the SWIR upconversion emission of small organic dyes, forming a AuNR-dye nanocomposite (NC). A maximal enhancement factor of ∼1320, contributed by both excitation and radiative decay rate enhancement, is achieved by varying the dye-to-AuNR ratio. In addition, the upconversion emission intensity of both free dyes and AuNR-dye NCs depends linearly on the excitation power, indicating that the upconversion emission mechanism remains unchanged upon enhancement, and it involves one-photon absorption. Moreover, the SWIR upconversion emission shows a significantly higher signal contrast than downconversion emission in the same emission window in a nonscattering medium. Finally, we apply the surface plasmon enhanced SWIR upconversion fluorescence for in vivo imaging of ovarian cancer, demonstrating high image contrast and low required dosage due to the suppressed autofluorescence.

Peptide-Based Cancer Vaccine Delivery via the STINGΔTM-cGAMP Complex.

With the advent of bioinformatic tools in efficiently predicting neo-antigens, peptide vaccines have gained tremendous attention in cancer immunotherapy. However, the delivery of peptide vaccines remains a major challenge, primarily due to ineffective transport to lymph nodes and low immunogenicity. Here, a strategy for peptide vaccine delivery is reported by first fusing the peptide to the cytosolic domain of the stimulator of interferon genes protein (STINGΔTM), then complexing the peptide-STINGΔTM protein with STING agonist 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The process results in the formation of self-assembled cGAMP-peptide-STINGΔTM tetramers, which enables efficient lymphatic trafficking of the peptide. Moreover, the cGAMP-STINGΔTM complex acts not only as a protein carrier for the peptide, but also as a potent adjuvant capable of triggering STING signaling independent of endogenous STING protein-an especially important attribute considering that certain cancer cells epigenetically silence their endogenous STING expression. With model antigen SIINFEKL, it is demonstrated that the platform elicits effective STING signaling in vitro, draining lymph node targeting in vivo, effective T cell priming in vivo as well as antitumoral immune response in a mouse colon carcinoma model, providing a versatile solution to the challenges faced in peptide vaccine delivery.

In vitro STING Activation with the cGAMP-STINGΔTM Signaling Complex.

Activating the STING (stimulator of interferon genes) signaling pathway via administration of STING agonist cyclic GMP-AMP (cGAMP) has shown great promise in cancer immunotherapy. While state-of-the-art approaches have predominantly focused on the encapsulation of cGAMP into liposomes or polymersomes for cellular delivery, we discovered that the recombinant STING protein lacking the transmembrane domain (STINGΔTM) could be used as a functional carrier for cGAMP delivery and elicit type I IFN expression in STING-deficient cell lines. Using this approach, we generated anti-tumoral immunity in mouse melanoma and colon cancer models, providing a potential translatable platform for STING agonist-based immunotherapy. Here, we report the detailed in vitro STING activation protocols with cGAMP-STINGΔTM complex to assist researchers in further development of this approach. This protocol can also be easily expanded to other applications related to STING activation, such as control of various types of infections.

Surface Plasmon-Enhanced Short-Wave Infrared Fluorescence for Detecting Sub-Millimeter-Sized Tumors.

Short-wave infrared (SWIR, 900-1700 nm) enables in vivo imaging with high spatiotemporal resolution and penetration depth due to the reduced tissue autofluorescence and decreased photon scattering at long wavelengths. Although small organic SWIR dye molecules have excellent biocompatibility, they have been rarely exploited as compared to their inorganic counterparts, mainly due to their low quantum yield. To increase their brightness, in this work, the SWIR dye molecules are placed in close proximity to gold nanorods (AuNRs) for surface plasmon-enhanced emission. The fluorescence enhancement is optimized by controlling the dye-to-AuNR number ratio and up to ≈45-fold enhancement factor is achieved. In addition, the results indicate that the highest dye-to-AuNR number ratio gives the highest emission intensity per weight and this is used for synthesizing SWIR imaging probes using layer-by-layer (LbL) technique with polymer coating protection. Then, the SWIR imaging probes are applied for in vivo imaging of ovarian cancer and the surface coating effect on intratumor distribution of the imaging probes is investigated in two orthotopic ovarian cancer models. Lastly, it is demonstrated that the plasmon-enhanced SWIR imaging probe has great potential for fluorescence imaging-guided surgery by showing its capability to detect sub-millimeter-sized tumors.

Temporal release of a three-component protein subunit vaccine from polymer multilayers.

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.

Self-assembled cGAMP-STINGΔTM signaling complex as a bioinspired platform for cGAMP delivery.

The stimulator of interferon (IFN) genes (STING) pathway constitutes a highly important part of immune responses against various cancers and infections. Consequently, administration of STING agonists such as cyclic GMP-AMP (cGAMP) has been identified as a promising approach to target these diseases. In cancer cells, STING signaling is frequently impaired by epigenetic silencing of STING; hence, conventional delivery of only its agonist cGAMP may be insufficient to trigger STING signaling. In this work, while expression of STING lacking the transmembrane (TM) domain is known to be unresponsive to STING agonists and is dominant negative when coexpressed with the full-length STING inside cells, we observed that the recombinant TM-deficient STING protein complexed with cGAMP could effectively trigger STING signaling when delivered in vitro and in vivo, including in STING-deficient cell lines. Thus, this bioinspired method using TM-deficient STING may present a universally applicable platform for cGAMP delivery.

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme.

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.

Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination.

The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has proven to be a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15-90 min) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate- b-methacrylic acid) (PDM), requiring only 1 min skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 µg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features, rapid administration and the ability to elicit a robust immune response, can potentially enable a broad application of microneedle-based vaccination technologies.

Structurally Programmed Assembly of Translation Initiation Nanoplex for Superior mRNA Delivery.

Messenger RNA (mRNA) represents a promising class of nucleic-acid-based therapeutics. While numerous nanocarriers have been developed for mRNA delivery, the inherent labile nature of mRNA results in a very low transfection efficiency and poor expression of desired protein. Here we preassemble the mRNA translation initiation structure through an inherent molecular recognition between 7-methylguanosine (m7G)-capped mRNA and eukaryotic initiation factor 4E (eIF4E) protein to form ribonucleoproteins (RNPs), thereby mimicking the first step of protein synthesis inside cells. Subsequent electrostatic stabilization of RNPs with structurally tunable cationic carriers leads to nanosized complexes (nanoplexes), which elicit high levels of mRNA transfection in different cell types by enhancing intracellular mRNA stability and protein synthesis. By investigating a family of synthetic polypeptides bearing different side group arrangements of cationic charge, we find that the molecular structure modulates the nanoscale distance between the mRNA strand and the eIF4E protein inside the nanoplex, which directly impacts the enhancement of mRNA transfection. To demonstrate the biomedical potential of this approach, we use this approach to introduce mRNA/eIF4E nanoplexes to murine dendritic cells, resulting in increased activation of cytotoxic CD8 T cells ex vivo. More importantly, eIF4E enhances gene expression in lungs following a systemic delivery of luciferase mRNA/eIF4E in mice. Collectively, this bioinspired molecular assembly method could lead to a new paradigm of gene delivery.

Synthetic Lift-off Polymer beneath Layer-by-Layer Films for Surface-Mediated Drug Delivery.

A broad range of biomaterials coatings and thin film drug delivery systems require a strategy for the immobilization, retention, and release of coatings from surfaces such as patches, inserts, and microneedles under physiological conditions. Here we report a polymer designed to provide a dynamic surface, one that first functions as a platform for electrostatic thin film assembly and releases the film once in an in vivo environment. Atom transfer radical polymerization (ATRP) was used to synthesize this polymer poly(o-nitrobenzyl-methacrylate-co-hydroxyethyl-methacrylate-co-poly(ethylene-glycol)-methacrylate) (PNHP), embedded beneath multilayered polyelectrolyte films. Such a base layer is designed to photochemically pattern negative charge onto a solid substrate, assist deposition of smooth layer-by-layer (LbL) polyelectrolyte in mildly acidic buffers and rapidly dissolve at physiological pH, thus lifting off the LbL films. To explore potential uses in the biomedical field, a lysozyme (Lys)/poly(acrylic acid) (PAA) multilayer film was developed on PNHP-coated silicon wafers to construct prototype antimicrobial shunts. Film thickness was shown to grow exponentially with increasing deposition cycles, and effective drug loading and in vitro release was confirmed by the dose-dependent inhibition of Escherichia coli (E. coli) growth. The efficacy of this approach is further demonstrated in LbL-coated microscale needle arrays ultimately of interest for vaccine applications. Using PNHP as a photoresist, LbL films were confined to the tips of the microneedles, which circumvented drug waste at the patch base. Subsequent confocal images confirmed rapid LbL film implantation of PNHP at microneedle penetration sites on mouse skin. Furthermore, in human skin biopsies, we achieved efficient immune activation demonstrated by a rapid uptake of vaccine adjuvant from microneedle-delivered PNHP LbL film in up to 37% of antigen-presenting cells (APC), providing an unprecedented LbL microneedle platform for human vaccination.