Development of an Accelerated Rotator-based Drug Release Method for the Evaluation of Bupivacaine Multivesicular Liposomes.

PURPOSE: A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. METHODS: Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24 h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. RESULTS: The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24 h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. CONCLUSIONS: An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.

Synthetic high-density lipoprotein (sHDL): a bioinspired nanotherapeutics for managing periapical bone inflammation.

Apical periodontitis (AP) is a dental-driven condition caused by pathogens and their toxins infecting the inner portion of the tooth (i.e., dental pulp tissue), resulting in inflammation and apical bone resorption affecting 50% of the worldwide population, with more than 15 million root canals performed annually in the United States. Current treatment involves cleaning and decontaminating the infected tissue with chemo-mechanical approaches and materials introduced years ago, such as calcium hydroxide, zinc oxide-eugenol, or even formalin products. Here, we present, for the first time, a nanotherapeutics based on using synthetic high-density lipoprotein (sHDL) as an innovative and safe strategy to manage dental bone inflammation. sHDL application in concentrations ranging from 25 µg to 100 µg/mL decreases nuclear factor Kappa B (NF-κB) activation promoted by an inflammatory stimulus (lipopolysaccharide, LPS). Moreover, sHDL at 500 µg/mL concentration markedly decreases in vitro osteoclastogenesis (P < 0.001), and inhibits IL-1α (P = 0.027), TNF-α (P = 0.004), and IL-6 (P < 0.001) production in an inflammatory state. Notably, sHDL strongly dampens the Toll-Like Receptor signaling pathway facing LPS stimulation, mainly by downregulating at least 3-fold the pro-inflammatory genes, such as Il1b, Il1a, Il6, Ptgs2, and Tnf. In vivo, the lipoprotein nanoparticle applied after NaOCl reduced bone resorption volume to (1.3 ± 0.05) mm3 and attenuated the inflammatory reaction after treatment to (1 090 ± 184) cells compared to non-treated animals that had (2.9 ± 0.6) mm3 (P = 0.012 3) and (2 443 ± 931) cells (P = 0.004), thus highlighting its promising clinical potential as an alternative therapeutic for managing dental bone inflammation.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Phospholipids impact the protective effects of HDL-mimetic nanodiscs against lipopolysaccharide-induced inflammation.

Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment.

Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature.

Robust anti-tumor T cell response with efficient intratumoral infiltration by nanodisc cancer immunotherapy.

Potent anti-tumor T cell response and efficient intratumoral T cell infiltration are the major challenges for therapeutic cancer vaccines. To address these issues, a nano-vaccine system has been designed to promote anti-tumor T cell responses, and intratumoral infiltration was examined in various murine tumor models. Subcutaneous vaccination with nanodiscs carrying human papillomavirus (HPV)-16 E7 antigen elicits as high as ~32% E7-specific CD8 α + T cell responses in circulation, representing a 29-fold improvement over the soluble peptide vaccination. Importantly, nanodisc vaccination also promotes robust intratumoral T cell infiltration and eliminates HPV16 E6/E7-expressing TC-1 tumors at mucosal sites, including lungs, inner lip, and intravaginal tissues. In a benchmark study with a live Listeria vaccine combined with anti-PD-1 IgG, nanodiscs plus anti-PD-1 immune checkpoint blockade elicits comparable levels of T cell responses with anti-tumor efficacy. Furthermore, compared with Complete Freund's Adjuvant combined with tetanus toxoid, nanodisc vaccination in HLA-A02 mice generates >200-fold stronger IFN-γ+ T cell responses against a neoantigen from an HLA-A02 melanoma patient. Overall, these results show that the nanodisc system is a promising cancer vaccine platform for inducing anti-tumor T cell responses.