Lipid Nanoparticle with 1,2-Di-O-octadecenyl-3-trimethylammonium-propane as a Component Lipid Confers Potent Responses of Th1 Cells and Antibody against Vaccine Antigen.

Adjuvants are effective tools to enhance vaccine efficacy and control the type of immune responses such as antibody and T helper 1 (Th1)- or Th2-type responses. Several studies suggest that interferon (IFN)-γ-producing Th1 cells play a significant role against infections caused by intracellular bacteria and viruses; however, only a few adjuvants can induce a strong Th1-type immune response. Recently, several studies have shown that lipid nanoparticles (LNPs) can be used as vaccine adjuvants and that each LNP has a different adjuvant activity. In this study, we screened LNPs to develop an adjuvant that can induce Th1 cells and antibodies using a conventional influenza split vaccine (SV) as an antigen in mice. We observed that LNP with 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) as a component lipid (DOTMA-LNP) elicited robust SV-specific IgG1 and IgG2 responses compared with SV alone in mice and was as efficient as SV adjuvanted with other adjuvants in mice. Furthermore, DOTMA-LNPs induced robust IFN-γ-producing Th1 cells without inflammatory responses compared to those of other adjuvants, which conferred strong cross-protection in mice. We also demonstrated the high versatility of DOTMA-LNP as a Th1 cell-inducing vaccine adjuvant using vaccine antigens derived from severe acute respiratory syndrome coronavirus 2 and Streptococcus pneumoniae. Our findings suggest the potential of DOTMA-LNP as a safe and effective Th1 cell-inducing adjuvant and show that LNP formulations are potentially potent adjuvants to enhance the effectiveness of other subunit vaccines.

Brain Delivery of Cisplatin Using Microbubbles in Combination with Ultrasound as an Effective Therapy for Glioblastoma.

Glioblastoma is a highly invasive and fatal disease. Temozolomide, a blood-brain barrier (BBB)-penetrant therapeutic agent currently used for glioblastoma, does not exhibit sufficient therapeutic effect. Cisplatin (CDDP), a versatile anticancer drug, is not considered a therapeutic option for glioblastoma due to its low BBB permeability. We previously investigated the utility of microbubbles (MBs) in combination with ultrasound (US) in promoting BBB permeability and reported the efficacy of drug delivery to the brain using a minimally invasive approach. This study aimed to evaluate the feasibility of CDDP delivery to the brain using the combination of MBs and US for the treatment of glioblastoma. We used mice that were implanted with glioma-261 GFP-Luc cells expressing luciferase as the glioblastoma model. In this model, after tumor inoculation, the BBB opening was induced using MBs and US, and CDDP was simultaneously administered. We found that the CDDP concentrations were higher at the glioblastoma site where the US was applied, although CDDP normally cannot pass through the BBB. Furthermore, the survival was longer in mice treated with CDDP delivered via MBs and US than in those treated with CDDP alone or those that were left untreated. These results suggest that the combination of MBs and US is an effective antitumor drug delivery system based on BBB opening in glioblastoma therapy.

[Development of Microbubbles for Theranostics and Its Application in Brain Targeted Drug Delivery].

Theranostics, a new medical term that combines therapeutics and diagnostics is considered an ideal system for medical care. Ultrasound is considered one of the most reasonable energies for the development of theranostics. Additionally, microbubbles, which are ultrasound contrast agents, have received considerable attention for their effectiveness in diagnosis and therapy. Microbubbles are composed of an inner gas and an outer shell composed of proteins or phospholipids. Under ultrasound exposure, the oscillation or collapse of microbubbles is induced depending on the intensity of the ultrasound. These mechanical effects are important for imaging, drug delivery, and ablation therapies. Therefore, it is essential that microbubbles reach the targeted site and induce mechanical effects to achieve effective and efficient diagnosis and therapy. We have previously developed novel microbubbles with high stability by optimizing the outer shell composition. Recently, microbubbles containing distearoylphosphatidyl glycerol showed high stability and prolonged circulation in the blood. These novel microbubbles may be useful for diagnosis and therapy. The combination of microbubbles and ultrasound has received considerable attention for brain-targeted drug delivery applications. We examined whether microbubbles can be used for brain-targeted drug delivery and evaluated the effect of the encapsulated gas on drug delivery. Thus, novel microbubbles combined with ultrasound can deliver molecules to the brain. Microbubbles containing perfluoropropane or perfluorobutane could efficiently deliver molecules to the brain. The novel microbubbles have long-circulating properties in the blood and could deliver molecules to the brain. The combination of novel microbubbles and ultrasound would contribute to the development of efficient thranostic systems.

Viability variation of T-cells under ultrasound exposure according to adhesion condition with bubbles.

PURPOSE: Although cellular immunotherapy is expected as a new cancer treatment, its therapeutic efficiency is limited in solid tumors, because most cells return to the bloodstream rather than adhere to the target site. Therefore, we are motivated to develop a technique to concentrate the cells in the blood flow using active control of bubble-surrounded cells under ultrasound exposure considering both aspects of cell controllability and viability. METHODS: We prepared a lipid bubble conjugating ligand to adhere to the surface of the T-cells. First, we evaluated the cell controllability by retaining the cells on a wall of an artificial blood vessel through continuous ultrasound exposure. Next, we investigated the cell viability under ultrasound exposure in a suspension with various bubble concentrations. RESULTS: We estimated the concentration of bubbles when the adhesion to the cell surface was saturated. Then, we evaluated the cell viability with various conditions of ultrasound exposure and bubble concentrations. However, it was confirmed that cell damage occurred under conditions that achieved proper control of the cells. Therefore, we exposed the cells to burst waves to reduce the applied ultrasound intensity. Consequently, the significant increase in cell viability was confirmed to be inversely proportional to the duty ratio. CONCLUSION: To retain cells on a vessel wall, determining the appropriate ultrasound condition including sound pressure and waveform is important to maintain cell viability.

Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives.

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized ß-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme ß-glucuronidase. To trigger the cell release of ß-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of ß-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact ß-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer.

Focused ultrasound/microbubbles-assisted BBB opening enhances LNP-mediated mRNA delivery to brain.

Messenger RNA (mRNA) medicine has become a new therapeutic approach owing to the progress in mRNA delivery technology, especially with lipid nanoparticles (LNP). However, mRNA encapsulated-LNP (mRNA-LNP) cannot spontaneously cross the blood-brain barrier (BBB) which prevents the expression of foreign proteins in the brain. Microbubble-assisted focused ultrasound (FUS) BBB opening is an emerging technology that can transiently enhance BBB permeability. In this study, FUS/microbubble-assisted BBB opening was investigated for the intravenous delivery of mRNA-LNP to the brain. The intensity of FUS irradiation was optimized to 1.5 kW/cm2, at which BBB opening occurred efficiently without hemorrhage or edema. Exogenous protein (luciferase) expression by mRNA-LNP, specifically at the FUS-irradiated side of the brain, occurred only when FUS and microbubbles were applied. This exogenous protein expression was faster but shorter than that of plasmid DNA delivery. Furthermore, foreign protein expression was observed in the microglia, along with CD31-positive endothelial cells, whereas no expression was observed in astrocytes or neurons. These results support the addition of mRNA-LNP to the lineup of nanoparticles delivered by BBB opening.

Ultrasound and microbubble-mediated drug delivery and immunotherapy.

Ultrasound induces the oscillation and collapse of microbubbles such as those of an ultrasound contrast agent, where these behaviors generate mechanical and thermal effects on cells and tissues. These, in turn, induce biological responses in cells and tissues, such as cellular signaling, endocytosis, or cell death. These physiological effects have been used for therapeutic purposes. Most pharmaceutical agents need to pass through the blood vessel walls and reach the parenchyma cells to produce therapeutic effects in drug delivery. Therefore, the blood vessel walls act as an obstacle to drug delivery. The combination of ultrasound and microbubbles is a promising strategy to enhance vascular permeability, improving drug transport from blood to tissues. This combination has also been applied to gene and protein delivery, such as cytokines and antigens for immunotherapy. Immunotherapy, in particular, is an attractive technique for cancer treatment as it induces a cancer cell-specific response. However, sufficient anti-tumor effects have not been achieved with the conventional cancer immunotherapy. Recently, new therapies based on immunomodulation with immune checkpoint inhibitors have been reported. Immunomodulation can be regarded as a new strategy for cancer immunotherapy. It was also reported that mechanical and thermal effects induced by the combination of ultrasound and microbubbles could suppress tumor growth by promoting the cancer-immunity cycle via immunomodulation in the tumor microenvironment. In this review, we provide an overview of the application of ultrasound and microbubble combination for drug delivery and activation of the immune system in the microenvironment of tumor tissue.

Ultrasound image-guided gene delivery using three-dimensional diagnostic ultrasound and lipid-based microbubbles.

Gene therapy is a promising technology for genetic and intractable diseases. Drug delivery carriers or systems for genes and nucleic acids have been studied to improve transfection efficiency and achieve sufficient therapeutic effects. Ultrasound (US) and microbubbles have also been combined for use in gene delivery. To establish a clinically effective gene delivery system, exposing the target tissues to US is important. The three-dimensional (3D) diagnostic probe can three-dimensionally scan the tissue with mechanical regulation, and homogenous US exposure to the targeted tissue can be expected. However, the feasibility of therapeutically applying 3D probes has not been evaluated, especially gene delivery. In this study, we evaluated the characteristics of a 3D probe and lipid-based microbubbles (LB) for gene delivery and determined whether the 3D probe in the diagnostic US device could be used for efficient gene delivery to the targeted tissue using a mouse model. The 3D probe RSP6-16 with LB delivered plasmid DNA (pDNA) to the kidney after systemic injection with luciferase activity similar to that of probes used in previously studies. No toxicity was observed after treatment and, therefore, the combined 3D probe and LB would deliver genes to targeted tissue safely and efficiently.

Feasibility study of novel nanoparticles derived from Glycyrrhizae radix as vaccine adjuvant for cancer immunotherapy.

Aims: The feasibility of using nanoparticles derived from Glycyrrhizae radix extract (Glycyrrhiza NPs) as a vaccine adjuvant for cancer immunotherapy was evaluated. Methods: C57BL/6J mice were immunized with ovalbumin (OVA) and Glycyrrhiza NPs. After immunization, splenocytes were incubated with the H-2Kb epitope peptide of OVA (SL8) and the production of IFN-γ was evaluated. Moreover, an OVA-expressing lymphoma cell line (E.G7-OVA cells) was inoculated into mice after immunization to evaluate the antitumor effect. Results: The immunization of OVA with Glycyrrhiza NPs induced IFN-γ production and completely rejected E.G7-OVA cells. Conclusion: Glycyrrhiza NPs could prime antigen-specific CD8+ T-cells resulting in antitumor effects. Therefore, Glycyrrhiza NPs can be an effective vaccine adjuvant for cancer immunotherapy.

Glycyrrhizae radix is a medical plant that contains anti-inflammatory compounds such as glycyrrhizin. Nanoparticles (NPs) derived from Glycyrrhizae radix extract induced the production of proinflammatory cytokines. Therefore, these NPs could be used as a vaccine adjuvant. Here, a feasibility study on the use of Glycyrrhiza NPs as a vaccine adjuvant in cancer immunotherapy is reported. T-cell responses and antitumor effects were evaluated after the immunization of ovalbumin (OVA) with Glycyrrhiza NPs. The immunization of OVA with Glycyrrhiza NPs effectively induced OVA-specific T-cells and completely rejected OVA-expressing tumor cells. Therefore, Glycyrrhiza NPs could induce antitumor immunity and be an effective vaccine adjuvant in cancer immunotherapy.

Enhanced Vascular Permeability by Microbubbles and Ultrasound in Drug Delivery.

Ultrasound and microbubbles, an ultrasound contrast agent, have recently increased attention to developing novel drug delivery systems. Ultrasound exposure can induce mechanical effects derived from microbubbles behaviors such as an expansion, contraction, and collapse depending on ultrasound conditions. These mechanical effects induce several biological effects, including enhancement of vascular permeability. For drug delivery, one promising approach is enhancing vascular permeability using ultrasound and microbubbles, resulting in improved drug transport to targeted tissues. This approach is applied to several tissues and drugs to cure diseases. This review describes the enhancement of vascular permeability by ultrasound and microbubbles and its therapeutic application, including our recent study. We also discuss the current situation of the field and its potential future perspectives.

Enhanced antitumor activity of combined lipid bubble ultrasound and anticancer drugs in gynecological cervical cancers.

Chemotherapy plays an important role in the treatment of patients with gynecological cancers. Delivering anticancer drugs effectively to tumor cells with just few side effects is key in cancer treatment. Lipid bubbles (LB) are compounds that increase the vascular permeability of the tumor under diagnostic ultrasound (US) exposure and enable the effective transport of drugs to tumor cells. The aim of our study was to establish a novel drug delivery technique for chemotherapy and to identify the most effective anticancer drugs for the bubble US-mediated drug delivery system (BUS-DDS) in gynecological cancer treatments. We constructed xenograft models using cervical cancer (HeLa) and uterine endometrial cancer (HEC1B) cell lines. Lipid bubbles were injected i.v., combined with either cisplatin (CDDP), pegylated liposomal doxorubicin (PLD), or bevacizumab, and US was applied to the tumor. We compared the enhanced chemotherapeutic effects of these drugs and determined the optimal drugs for BUS-DDS. Tumor volume reduction of HeLa and HEC1B xenografts following cisplatin treatment was significantly enhanced by BUS-DDS. Both CDDP and PLD significantly enhanced the antitumor effects of BUS-DDS in HeLa tumors; however, volume reduction by BUS-DDS was insignificant when combined with bevacizumab, a humanized anti-vascular endothelial growth factor mAb. The BUS-DDS did not cause any severe adverse events and significantly enhanced the antitumor effects of cytotoxic drugs. The effects of bevacizumab, which were not as dose-dependent as those of the two drugs used prior, were minimal. Our data suggest that BUS-DDS technology might help achieve "reinforced targeting" in the treatment of gynecological cancers.

Lipid bubbles combined with low-intensity ultrasound enhance the intratumoral accumulation and antitumor effect of pegylated liposomal doxorubicin in vivo.

Pegylated liposomal doxorubicin (PLD) is a representative nanomedicine that has improved tumor selectivity and safety profile. However, the therapeutic superiority of PLD over conventional doxorubicin has been reported to be insignificant in clinical medicine. Combination treatment with microbubbles and ultrasound (US) is a promising strategy for enhancing the antitumor effects of chemotherapeutics by improving drug delivery. Recently, several preclinical studies have shown the drug delivery potential of lipid bubbles (LBs), newly developed monolayer microbubbles, in combination with low-intensity US (LIUS). This study aimed to elucidate whether the combined use of LBs and LIUS enhanced the intratumoral accumulation and antitumor effect of PLD in syngeneic mouse tumor models. Contrast-enhanced US imaging using LBs showed a significant decrease in contrast enhancement after LIUS, indicating that LIUS exposure induced the destruction of LBs in the tumor tissue. A quantitative evaluation revealed that the combined use of LBs and LIUS improved the intratumoral accumulation of PLD. Furthermore, tumor growth was inhibited by combined treatment with PLD, LBs, and LIUS. Therefore, the combined use of LBs and LIUS enhanced the antitumor effect of PLD by increasing its accumulation in the tumor tissue. In conclusion, the present study provides important evidence that the combination of LBs and LIUS is an effective method for enhancing the intratumoral delivery and antitumor effect of PLD in vivo.

Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression.

Glutathione (GSH) is an important antioxidant that plays a critical role in neuroprotection. GSH depletion in neurons induces oxidative stress and thereby promotes neuronal damage, which in turn is regarded as a hallmark of the early stage of neurodegenerative diseases. The neuronal GSH level is mainly regulated by cysteine transporter EAAC1 and its inhibitor, GTRAP3-18. In this study, we found that the GTRAP3-18 level was increased by up-regulation of the microRNA miR-96-5p, which was found to decrease EAAC1 levels in our previous study. Since the 3'-UTR region of GTRAP3-18 lacks the consensus sequence for miR-96-5p, an unidentified protein should be responsible for the intermediate regulation of GTRAP3-18 expression by miR-96-5p. Here, we discovered that RNA-binding protein NOVA1 functions as an intermediate protein for GTRAP3-18 expression via miR-96-5p. Moreover, we show that intra-arterial injection of a miR-96-5p-inhibiting nucleic acid to living mice by a drug delivery system using microbubbles and ultrasound decreased the level of GTRAP3-18 via NOVA1 and increased the levels of EAAC1 and GSH in the dentate gyrus of the hippocampus. These findings suggest that the delivery of a miR-96-5p inhibitor to the brain would efficiently increase the neuroprotective activity by increasing GSH levels via EAAC1, GTRAP3-18 and NOVA1.

Effect of lipid shell composition in DSPG-based microbubbles on blood flow imaging with ultrasonography.

Diagnostic ultrasound is non-invasive and provides real-time imaging. Microbubbles (MBs) are ultrasound contrast agents used to observe small blood flow, such as tumor tissue. However, MBs have short blood flow imaging time. This study developed lipid-based microbubbles (LMBs) with longer blood flow imaging time by focusing on their shell composition. Liposome research reported that addition 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) to the lipid composition enhances liposome membrane stability. Therefore, we introduced DSPG at different ratios into the LMBs lipid shell. Results showed that the lipid shell composition of MBs affects stability in vivo. 60% DSPG-containing LMBs (DSPG60-LMBs) have sustained blood flow imaging time compared with LMBs, which have other DSPG ratios, Sonazoid® and SonoVue®. DSPG60-LMBs also showed less uptake into the liver compared with Sonazoid®. Therefore, DSPG60-LMBs can have long blood flow imaging time and can be effective diagnostic agents in ultrasound imaging.

Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses.

Vaccination is a critical and reliable strategy for controlling the spread of influenza viruses in populations. Conventional seasonal split vaccines (SVs) for influenza evoke weaker immune responses than other types of vaccines, such as inactivated whole-virion vaccines, although SVs are highly safe compared to other types. Here, we assessed the potential of the lipid nanoparticle (LNP) we developed as an adjuvant for conventional influenza SV as an antigen in mice. The LNP did not induce the production of cytokines such as interleukin-6 (IL-6) and IL-12 p40 by dendritic cells or the expression of co-stimulatory molecules on these cells in vitro. In contrast, an SV adjuvanted with LNP improved SV-specific IgG1 and IgG2 responses and the Th1 response compared to the SV alone in mice. In addition, SV adjuvanted with an LNP gave superior protection against the influenza virus challenge over the SV alone and was as effective as SV adjuvanted with aluminum salts in mice. The LNP did not provoke inflammatory responses such as inflammatory cytokine production and inflammatory immune cell infiltration in mice, whereas aluminum salts induced inflammatory responses. These results suggest the potential of the LNP as an adjuvant without inflammatory responses for influenza SVs. Our strategy should be useful for developing influenza vaccines with enhanced efficacy and safety.

A Pilot Study on Efficacy of Lipid Bubbles for Theranostics in Dogs with Tumors.

The combined administration of microbubbles and ultrasound (US) is a promising strategy for theranostics, i.e., a combination of therapeutics and diagnostics. Lipid bubbles (LBs), which are experimental theranostic microbubbles, have demonstrated efficacy in vitro and in vivo for both contrast imaging and drug delivery in combination with US irradiation. To evaluate the clinical efficacy of LBs in combination with US in large animals, we performed a series of experiments, including clinical studies in dogs. First, contrast-enhanced ultrasonography using LBs (LB-CEUS) was performed on the livers of six healthy Beagles. The hepatic portal vein and liver tissue were enhanced; no adverse reactions were observed. Second, LB-CEUS was applied clinically to 21 dogs with focal liver lesions. The sensitivity and specificity were 100.0% and 83.3%, respectively. These results suggested that LB-CEUS could be used safely for diagnosis, with high accuracy. Finally, LBs were administered in combination with therapeutic US to three dogs with an anatomically unresectable solid tumor in the perianal and cervical region to determine the enhancement of the chemotherapeutic effect of liposomal doxorubicin; a notable reduction in tumor volume was observed. These findings indicate that LBs have potential for both therapeutic and diagnostic applications in dogs in combination with US irradiation.

Lipid-based microbubbles and ultrasound for therapeutic application.

Microbubbles with diagnostic ultrasound have had a long history of use in the medical field. In recent years, the therapeutic application of the combination of microbubbles and ultrasound, called sonoporation, has received increased attention as microbubble oscillation or collapse close to various barriers in the body was recognized to potentially open those barriers, increasing drug transport across them. In this review, we aimed to describe the development of lipid-stabilized microbubbles equipped with functions, such as long circulation and drug loading, and the therapeutic application of sonoporation for tumor-targeted therapy, brain-targeted therapy, and immunotherapy. We also attempted to discuss the current status of the field and potential future developments.

Characterization of Brain-Targeted Drug Delivery Enhanced by a Combination of Lipid-Based Microbubbles and Non-Focused Ultrasound.

The combination of focused ultrasound (FUS) and microbubbles, an ultrasound (US) contrast agent, has attracted much attention for its ability to open the blood brain barrier (BBB) and deliver drugs to the brain parenchyma. FUS can concentrate US energy in a restricted space, whereas non-focused US can affect a wide area of tissue. Non-focused US is also promising for drug delivery to the brain and other tissues. We have previously developed lipid-based microbubbles (LBs), and demonstrated that non-focused US and LBs have potential for drug delivery to tumor tissues. In this study, to achieve efficient and safe brain-targeted drug delivery, we evaluated the characteristics of BBB opening using non-focused US and LBs. Our results indicated that LBs could induce BBB opening with non-focused US. US frequency and intensity affected the efficiency of BBB opening and brain damage, and showed that the dose of LBs was also related to the efficiency of BBB opening. Furthermore, the combination of non-focused US and LBs could deliver macromolecules at 2000 kDa to the brain, and the induction of BBB opening was found to be reversible. These results suggest that the combination of non-focused US and LBs has potential as a brain-targeted drug delivery system.

Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for ultrasound imaging and therapy.

Microbubble formulations have a long history for enhancement of ultrasound (US) imaging and recently also for therapeutic applications. Previously, a series of freeze-dried bubble formulations based on the lipids DSPC and DSPG were developed. Here, we have attempted to scale-up the production process for future more extensive studies. Bubbles were prepared by homogenization of a lipid dispersion in a perfluoropropane atmosphere in a medium size (300-500 mL) homogenizer and then freeze-dried for better storage stability. In total, 300 freeze-dried vials were prepared. The properties of the bubbles were similar to those previously prepared on a lab scale with the difference that they were slightly larger and also had a better stability. The re-entrapped gas concentration after re-constituted freeze-dried bubbles was 9.4 µL/µmol lipid. The re-entrapped rate was 72.3% of fresh bubble before freeze-drying (13.0 µL/µmol lipid). The half-life of US imaging signal of the re-constituted freeze-dried bubbles in water in vitro was shorter than that of the fresh bubbles (2.7 min vs. 3.3 min). A leak of Evans Blue, that binds to albumin, from mouse ear blood vessel was observed after combination of bubble and US irradiation of 1 MHz for 1 min. As a result of bubble vibration by US irradiation, vascular endothelial cell bond opened and Evans Blue leaked. Toxicity of bubble was tested in rats. No toxicity was found after a single injection in the dose range tested. No serious toxicity was seen after repeated injections (one daily injection during 15 days).

Lipid nanoparticles of Type-A CpG D35 suppress tumor growth by changing tumor immune-microenvironment and activate CD8 T cells in mice.

Type-A CpG oligodeoxynucleotides (ODNs), which have a natural phosphodiester backbone, is one of the highest IFN-α inducer from plasmacytoid dendritic cells (pDC) via Toll-like receptor 9 (TLR9)-dependent signaling. However, the in vivo application of Type-A CpG has been limited because the rapid degradation in vivo results in relatively weak biological effect compared to other Type-B, -C, and -P CpG ODNs, which have nuclease-resistant phosphorothioate backbones. To overcome this limitation, we developed lipid nanoparticles formulation containing a Type-A CpG ODN, D35 (D35LNP). When tested in a mouse tumor model, intratumoral and intravenous D35LNP administration significantly suppressed tumor growth in a CD8 T cell-dependent manner, whereas original D35 showed no efficacy. Tumor suppression was associated with Th1-related gene induction and activation of CD8 T cells in the tumor. The combination of D35LNP and an anti-PD-1 antibody increased the therapeutic efficacy. Importantly, the therapeutic schedule and dose of intravenous D35LNP did not induce apparent liver toxicity. These results suggested that D35LNP is a safe and effective immunostimulatory drug formulation for cancer immunotherapy.