Precision Nanovaccines for Potent Vaccination.

Compared with traditional vaccines, nanoparticulate vaccines are especially suitable for delivering antigens of proteins, peptides, and nucleic acids and facilitating lymph node targeting. Moreover, apart from improving pharmacokinetics and safety, nanoparticulate vaccines assist antigens and molecular adjuvants in crossing biological barriers, targeting immune organs and antigen-presenting cells (APC), controlled release, and cross-presentation. However, the process that stimulates and orchestrates the immune response is complicated, involving spatiotemporal interactions of multiple cell types, including APCs, B cells, T cells, and macrophages. The performance of nanoparticulate vaccines also depends on the microenvironments of the target organs or tissues in different populations. Therefore, it is necessary to develop precise nanoparticulate vaccines that accurately regulate vaccine immune response beyond simply improving pharmacokinetics. This Perspective summarizes and highlights the role of nanoparticulate vaccines with precise size, shape, surface charge, and spatial management of antigen or adjuvant for a precision vaccination in regulating the distribution, targeting, and immune response. It also discusses the importance of the rational design of nanoparticulate vaccines based on the anatomical and immunological microstructure of the target tissues. Moreover, the target delivery and controlled release of nanovaccines should be taken into consideration in designing vaccines for achieving precise immune responses. Additionally, it shows that the nanovaccines remodel the suppressed tumor environment and modulate various immune cell responses which are also essential.

A cocktail nanovaccine targeting key entry glycoproteins elicits high neutralizing antibody levels against EBV infection.

Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.

Multisubcellular organelle-targeting nanoparticle for synergistic chemotherapy and photodynamic/photothermal tumor therapy.

Background: The subcellular organelle-targeting strategy has attracted wide attention for a variety of reasons, including strong specificity, high accuracy, low dose administration and few side effects. It is an important and challenging task to explore the multisubcellular organelle-targeting strategy to achieve effective tumor treatment. Materials & methods: Using bovine serum albumin as a nanoreactor, BSA/Cu/NQ/IR780/DOX nanoparticles (NPs) were constructed via drug-induced protein self-assembly. Folic acid was then coupled to the surface of NPs to prepare folate receptor-targeted FA-BSA/Cu/NQ/IR780/DOX NPs. Results & conclusion: The FA-BSA/Cu/NQ/IR780/DOX NPs exhibit multifunctional properties, including multisubcellular organelle-targeting, induction of response release in the tumor microenvironment, fluorescence imaging capabilities and potential for synergistic chemotherapy and photodynamic/photothermal tumor therapy.

The subcellular organelle-targeting strategy has attracted wide attention for a variety of reasons, including strong specificity, high accuracy, low dose administration and few side effects. Previous research has been mostly restricted to one or two subcellular organelle therapies. Despite promising results, the impact of these studies is limited by the hostile conditions of lysosomes, drug efflux facilitated by P-glycoprotein (P-gp), and the expression of antiapoptotic factors, all of which undermine the effectiveness of the treatments. Therefore, it is an important and challenging task to explore the multisubcellular organelle-targeting strategy to achieve effective tumor treatment. Herein, a versatile nanoparticle was designed and constructed to target multiple subcellular organelles, respond to stimuli in the tumor microenvironment, enable fluorescence imaging and facilitate synergistic chemotherapy and photodynamic/photothermal tumor therapy.

Nanovaccines Fostering Tertiary Lymphoid Structure to Attack Mimicry Nasopharyngeal Carcinoma.

Tertiary lymphoid structures (TLSs) are formed in inflamed tissues, and recent studies demonstrated that the appearance of TLSs in tumor sites is associated with a good prognosis for tumor patients. However, the process of natural TLSs' formation was slow and uncontrollable. Herein, we developed a nanovaccine consisting of Epstein-Barr virus nuclear antigen 1 (EBNA1) and a bi-adjuvant of Mn2+ and cytosine-phosphate-guanine (CpG) formulated with tannic acid that significantly inhibited the development of mimicry nasopharyngeal carcinoma by fostering TLS formation. The nanovaccine activated LT-α and LT-ß pathways, subsequently enhancing the expression of downstream chemokines, CCL19/CCL21, CXCL10 and CXCL13, in the tumor microenvironment. In turn, normalized blood and lymph vessels were detected in the tumor tissues of the nanovaccine group, correlated with increased infiltration of lymphocytes. Especially, the proportion of the B220+ CD8+ T, which was produced via trogocytosis between T and B cells during activation of T cells, was increased in tumors of the nanovaccine group. Furthermore, the intratumoral effector memory T cells (Tem), CD45+, CD3+, CD8+, CD44+, and CD62L-, did not decrease after blocking the egress of T cells from tumor-draining lymph nodes by FTY-720. These results demonstrated that the nanovaccine can foster TLS formation, which thus enhances local immune responses significantly, delays tumor outgrowth, and prolongs the median survival time of murine models of mimicry nasopharyngeal carcinoma, demonstrating a promising strategy for nanovaccine development.

CCR7 Mediated Mimetic Dendritic Cell Vaccine Homing in Lymph Node for Head and Neck Squamous Cell Carcinoma Therapy.

Immunotherapy has been recognized as one of the most promising treatment strategies for head and neck squamous cell carcinoma (HNSCC). As a pioneering trend of immunotherapy, dendritic cell (DC) vaccines have displayed the ability to prime an immune response, while the insufficient immunogenicity and low lymph node (LN) targeting efficiency, resulted in an unsubstantiated therapeutic efficacy in clinical trials. Herein, a hybrid nanovaccine (Hy-M-Exo) is developed via fusing tumor-derived exosome (TEX) and dendritic cell membrane vesicle (DCMV). The hybrid nanovaccine inherited the key protein for lymphatic homing, CCR7, from DCMV and demonstrated an enhanced efficiency of LN targeting. Meanwhile, the reserved tumor antigens and endogenous danger signals in the hybrid nanovaccine activated antigen presenting cells (APCs) elicited a robust T-cell response. Moreover, the nanovaccine Hy-M-Exo displayed good therapeutic efficacy in a mouse model of HNSCC. These results indicated that Hy-M-Exo is of high clinical value to serve as a feasible strategy for antitumor immunotherapy.

Carrier-free subunit nanovaccine amplifies immune responses against tumors and viral infections.

Codelivering subunit antigens and Toll-like receptor (TLR) molecular adjuvants via nanocarriers can stimulate potent innate and specific immune responses. Simple and effective nanovaccines fabrication is crucial for application. However, most nanovaccines were fabricated by introducing additional delivery materials, increasing safety risk, cost and processing complexity. Herein, a carrier-free nanovaccine was facilely prepared using a TLR1/TLR2 adjuvant, Diprovocim, rich in benzene rings that could interact with aromatic residues in subunit antigens through π-π stacking without additional materials. The carrier-free nanovaccines with a narrow size distribution could target lymph nodes (LNs) after intravenous injection to mice. The carrier-free nanovaccines based on ovalbumin (OVA) can stimulate strong antibody titers and CD4+ and CD8+ T cell immune responses in mice, and it synergized with anti-PD1 showing a potent tumor suppression in B16F10-OVA tumor model of mice. Furthermore, the carrier-free nanovaccine with glycoprotein E (gE), a glycoprotein of the varicella-zoster virus (VZV), also showed potent humoral and cellular immune responses. Therefore, using subunit proteins to support Diprovocim by π-π stacking provides a new approach for the preparation and application of novel vaccines for tumor therapy and prevention of infectious diseases. STATEMENT OF SIGNIFICANCE: Codelivering subunit antigens and adjuvants via nanocarriers stimulate potent innate and specific immune responses. However, existing delivery materials for fabricating nanovaccines will inevitably increase the cost of preparation, controllability, process complexity and safety assessment. Therefore, this study easily prepared carrier-free nanovaccines using the benzene ring-rich TLR1/TLR2 adjuvant Diprovocim, which can interact with aromatic residues in subunit antigens via π-π stacking without additional materials. The carrier-free nanovaccines of OVA demonstrated a potent tumor inhibition in treating melanoma in combination with anti-PD1. And the nanovaccines of gE stimulated a strong antibody titer and cellular immune response for herpes zoster. Thus, the present study provides a new approach for the preparation of subunit vaccines to combat various cancers and virus infections.

Nanoadjuvants Actively targeting lymph node conduits and blocking tumor invasion in lymphatic vessels.

Great efforts have been made to manipulate nanoparticles (NPs) with a diameter of 10-100 nm to passively target lymph nodes (LNs) to magnitude anti-tumor activity of T cells. However, no attention has been paid to increasing the retention of NPs with active affinity in order to induce a prolonged release of antigens or molecular adjuvants in the LNs mattering the immune response. Here, we formulated two NPs encapsulated with imiquimod (IMQ), a TLR7/8 agonist, and paclitaxel (PTX) and further modified them with tannic acid (TA), respectively, to generate IMQ NP and PTX NP with a final diameter of approximately 40 nm. Attributing a strong affinity of TA molecules to the elastin of LN conduits, the TA modified IMQ NPs can bypass the gaps in the layer of lymphatic endothelial cells and enter the paracortex through the lymph node capsule-associated (LNC) conduits. Similarly, the TA modified PTX NPs increased delivery of PTX to the metastatic tumor site in LNs, where the tumor-associated antigens were released and presented by conduits-lining dendritic cells to activate T cells. Thus, the NPs with deposition to LN conduits showed excellent performance in preventing lymphovascular invasion of triple-negative breast cancer cells and lung metastasis thereafter. On the contrary, the NPs without TA flowed through the subcutaneous sinus existing LNs directly by efferent lymphatic vessels showing relatively poor therapeutic outcomes. This study reveals that TA may mediate the long retention of antigens and molecular adjuvants to be delivered to deep LNs for developing potent vaccination technology.

Combined effect of heat shock protein inhibitor geldanamycin and free radicals on photodynamic therapy of prostate cancer.

Prostate cancer is the most common malignancy and the second leading cause of cancer-induced death among men. Recently, photodynamic therapy (PDT) has attracted great attention in prostate cancer treatment because of its high accuracy and no trauma. However, the hypoxic microenvironment of the tumor severely reduces the therapeutic efficacy of oxygen-dependent PDT in prostate cancer, which hampers the generation of reactive oxygen species (ROS). In addition, the PDT process induces the overexpression of pro-survival and anti-apoptotic proteins, thereby reducing the efficacy of PDT. This study proposed a novel multifunctional nanosystem for the targeted delivery of indocyanine green (ICG), 2,2'-azobis[2-(2-imidazolinI-2-yl) propane] dihydrochloride (AIBI), and heat shock protein 90 (Hsp90) inhibitor geldanamycin (17-AAG). Under near-infrared light irradiation, the photothermal effect of ICG induces AIBI decomposition and releases oxygen-independent free radicals, which rescues the hindered ICG-mediated ROS generation. Moreover, 17-AAG reduces heat resistance by inhibiting Hsp90, thereby achieving mild hyperthermia. Simultaneously, the inhibition of Hsp90 can inhibit the overexpression of its client proteins such as anti-apoptotic proteins (survivin) and androgen receptor (AR), thereby improving the efficacy of PDT and inducing prostate cancer cell apoptosis. Results show that the nanosystem enhances PDT by combining free radicals and 17-AAG, exhibiting a good anticancer effect on prostate cancer cells but less toxicity on normal cells.

Impact of exogenous estradiol on task-based and resting-state neural signature during and after fear extinction in healthy women.

Fluctuations of endogenous estrogen modulates fear extinction, but the influence of exogenous estradiol is less studied. Moreover, little focus has been placed on the impact of estradiol on broad network connectivity beyond the fear extinction circuit. Here, we examined the effect of acute exogenous estradiol administration on fear extinction-induced brain activation, whole-brain functional connectivity (FC) during the fear extinction task and post-extinction resting-state. Ninety healthy women (57 using oral contraceptives [OC], 33 naturally cycling [NC]) were fear conditioned on day 1. They ingested an estradiol or placebo pill prior to extinction learning on day 2 (double-blind design). Extinction memory was assessed on day 3. Task-based functional MRI data were ascertained on days 2 and 3 and resting-state data were collected post-extinction on day 2 and pre-recall on day 3. Estradiol administration significantly modulated the neural signature associated with fear extinction learning and memory, consistent with prior studies. Importantly, estradiol administration induced significant changes in FC within multiple networks, including the default mode and somatomotor networks during extinction learning, post-extinction, and during extinction memory recall. Exploratory analyses revealed that estradiol impacted ventromedial prefrontal cortex (vmPFC) activation and FC differently in the NC and OC women. The data implicate a more diffused and significant effect of acute estradiol administration on multiple networks. Such an effect might be beneficial to modulating attention and conscious processes in addition to engaging neural processes associated with emotional learning and memory consolidation.

Assembly of multifunction dyes and heat shock protein 90 inhibitor coupled to bovine serum albumin in nanoparticles for multimodal photodynamic/photothermal/chemo-therapy.

The proangiogenic protein, survivin, is a client protein for heat shock protein 90 (Hsp-90), whose overexpression is induced by photodynamic therapy (PDT), leading to the inhibition of capase-9 and the blockage of apoptosis. The overexpression of Hsp-90 in cancer cells can rapidly acquire thermoresistance during photothermal therapy (PTT), leading to insufficient apoptosis, increased cell viability, and tumor recurrence. A potential approach to block the PTT-induced overexpression of Hsp-90 and the overexpression of survivin is developed by using an Hsp-90 inhibitor and anticancer agent, namely, geldanamycin (GM). These inhibitors also develop a mild-temperature PTT strategy to reach synergistic PDT and PTT efficiency. Thus, Cy7-SQ is designed by a covalent disulfide linkage between a photothermal agent (i.e., canine dye 7 [Cy7]) and a photosensitizer (i.e., squaraine dye [SQ]) for the improved photostability and thermal stability of Cy7 and SQ. The cleavage of the Cy7-SQ linkage by glutathione in a tumor microenvironment increases the efficiency of synergistic PDT and PTT. In the current study, bovine serum albumin (BSA)/Cy7-SQ/GM nanoparticles are developed through the self-assembly of BSA, Cy7-SQ, and GM to accelerate the apoptosis of cancer cells via near-infrared (NIR) laser irradiation, thus realizing Hsp-90-regulated synergistic PDT/PTT combined with chemotherapy.

Nanoscale photosensitizer with tumor-selective turn-on fluorescence and activatable photodynamic therapy treatment for COX-2 overexpressed cancer cells.

Effective targeting and in situ imaging-guided treatment are particularly important for accurate clinical photodynamic therapy (PDT) of malignant tumors. Herein, we propose a single molecule, named IMC-DAH-SQ, which possesses dual-targeting components, including structure-inherent targeting (SIT) and cyclooxygenase-2 (COX-2) targeting units, and controllable turn-on near infrared (NIR) fluorescence. Due to its amphiphilicity, IMC-DAH-SQ assembles into a nanoprobe with low background fluorescence. After incubation with tumor cells, the SIT and COX-2 recognition characteristics of IMC-DAH-SQ endow it with preferential tumor-targeting activity. The strong binding with overexpressed COX-2 can collapse the nanoprobe to monomers after accumulation in tumor cells, leading to turn-on NIR fluorescence that is completely different from normal cells. Additionally, benefiting from the single molecular model tactic, the nanoprobe has the advantages of simple synthesis without ever considering the loading rate and separation between the photosensitizer and targeting unit. Other favorite features, including superior biocompatibility, weak dark toxicity, and mitochondria enrichment capability, are implemented. All these traits not only afford nanoprobe precision tumor cell targeting capability but also provide promising imaging-guided antitumor therapy. We believe that the single molecular protocol will establish a novel strategy for simultaneous diagnosis and anticancer medicine treatment utilizing versatile but small compounds.

A novel fluorescent probe based on naphthalimide for imaging nitroreductase (NTR) in bacteria and cells.

A nitroreductase (NTR) responsive fluorescent probe, Na-NO2, comprising p-nitrobenzyl as the unique recognition group and 1,8-naphthalimide as fluorophore, was synthesized. Na-NO2 showed remarkable fluorescence "turn-on" signal in the presence of NTR under DMSO/H2O (1:19, v/v) buffered with PBS (pH = 7) solution in the presence of NADH (300 µM). Furthermore, the probe has a low detection limit down to 3.4 ng/mL and it is very sensitive towards the NTR in Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), normal and tumor cells such as HL-7702, HepG-2 and MCF-7.

Recent development in biodegradable nanovehicle delivery system-assisted immunotherapy.

Based on the stimulation of the patient's own innate and adaptive immunity, immunotherapy is used in tumor treatment, and in recent years, it has developed rapidly. To generate strong and long-lasting antitumor immune responses and to boost clinical efficiency, various nanovehicle delivery systems (NDSs) have been designed to achieve specific release and avoid premature leakage in the delivery process. However, most of them are nondegradable, and they are taken up by the liver and spleen. This process leads to the accumulation of these substances in the human body, raising long-term toxicity concerns and causing potential undesirable side effects. To solve this problem, biodegradable NDSs (BNDSs) have been developed. Once these substances reach their target, they can only be degraded under specific internal or external stimuli, such as enzymes, irradiation, temperature, redox potential, pH, or a combination of these stimuli. Therefore, they are quite significant for potential clinical applications. In this review article, we highlight the recent literature on the design and working mechanism of various BNDSs. According to their type and characteristics, BNDSs are categorized as follows: (1) polymers, (2) lipid based materials, (3) inorganic nanomaterials, (4) biomacromolecules, (5) hybrid materials and (6) other materials. The existing challenges and future prospects of these materials will also be discussed.