Liposomal PHD2 Inhibitors and the Enhanced Efficacy in Stabilizing HIF-1α

Prolyl hydroxylase domain-containing protein 2 (PHD2) inhibition, which stabilizes hypoxia-inducible factor (HIF)-1α and thus triggers adaptation responses to hypoxia in cells, has become an important therapeutic target. Despite the proven high potency, small-molecule PHD2 inhibitors such as IOX2 may require a nanoformulation for favorable biodistribution to reduce off-target toxicity. A liposome formulation for improving the pharmacokinetics of an encapsulated drug while allowing a targeted delivery is a viable option. This study aimed to develop an efficient loading method that can encapsulate IOX2 and other PHD2 inhibitors with similar pharmacophore features in nanosized liposomes. Driven by a transmembrane calcium acetate gradient, a nearly 100% remote loading efficiency of IOX2 into liposomes was achieved with an optimized extraliposomal solution. The electron microscopy imaging revealed that IOX2 formed nanoprecipitates inside the liposome’s interior compartments after loading. For drug efficacy, liposomal IOX2 outperformed the free drug in inducing the HIF-1α levels in cell experiments, especially when using a targeting ligand. This method also enabled two clinically used inhibitors—vadadustat and roxadustat—to be loaded into liposomes with a high encapsulation efficiency, indicating its generality to load other heterocyclic glycinamide PHD2 inhibitors. We believe that the liposome formulation of PHD2 inhibitors, particularly in conjunction with active targeting, would have therapeutic potential for treating more specifically localized disease lesions.

Topical Triamcinolone Acetonide-Loaded Liposome Formulation Used as an Adjuvant to Intravitreal Ranibizumab Therapy for Neovascular Age-Related Macular Degeneration

Novel strategies have been developed to reduce or avoid intravitreal injections (IVTs) of the antiangiogenic (ranibizumab (RBZ)) and anti-inflammatory (triamcinolone acetonide (TA)) agents used to treat vitreoretinal diseases. One of the strategies includes liposomes. This study evaluated the safety and efficacy of a topical triamcinolone-loaded liposome formulation (TALF) as an adjuvant to intravitreal RBZ therapy in treatment- naïve patients with neovascular age-related macular degeneration (nAMD). Subjects were randomly assigned to the RBZ-TALF or the RBZ-pro re nata (RBZ-PRN) groups. Patients from the RBZ-TALF group were instructed to apply TALF for 12 months after a single dose of RBZ. Patients from the RBZ-PRN group received three monthly RBZ-IVTs. Retreatment with RBZ was considered in the case of nAMD reactivation. Regarding safety, non-ocular abnormalities were observed during TALF therapy. Concerning efficacy, non-significant differences were identified in terms of visual acuity or central foveal thickness when the RBZ-PRN and RBZ-TALF groups were compared. It is worth noting that the average number of RBZ injections was significantly lower in the RBZ-TALF group (2.5 ± 1.4 vs. 6.1 ± 1.3 IVTs; p = 0.0004). Therefore, TALF used as an adjuvant to RBZ reduces the need for RBZ-IVT retreatment with optimal visual and anatomic results.

Liposomes Loaded with Unsaponifiable Matter from Amaranthus hypochondriacus as a Source of Squalene and Carrying Soybean Lunasin Inhibited Melanoma Cells

Amaranthus hypochondriacus is a source of molecules with reported health benefits such as antioxidant activity and cancer prevention. The objective of this research was to optimize the conditions for preparing a liposome formulation using amaranth unsaponifiable matter as a source of squalene in order to minimize the particle size and to maximize the encapsulation efficiency of liposomes for carrying and delivering soybean lunasin into melanoma cell lines. Amaranth oil was extracted using supercritical dioxide carbon extraction (55.2 MPa pressure, 80 °C temperature, solvent (CO2)-to-feed (oil) ratio of 20). The extracted oil from amaranth was used to obtain the unsaponifiable enriched content of squalene, which was incorporated into liposomes. A Box–Behnken response surface methodology design was used to optimize the liposome formulation containing the unsaponifiable matter, once liposomes were optimized. Soybean lunasin was loaded into the liposomes and tested on A-375 and B16-F10 melanoma cells. The squalene concentration in the extracted oil was 36.64 ± 0.64 g/ 100 g of oil. The particle size in liposomes was between 115.8 and 163.1 nm; the squalene encapsulation efficiency ranged from 33.14% to 76.08%. The optimized liposome formulation contained 15.27 mg of phospholipids and 1.1 mg of unsaponifiable matter. Cell viability was affected by the liposome formulation with a half-maximum inhibitory concentration (IC50) equivalent to 225 μM in B16-F10 and 215 μM in A-375. The liposomes formulated with lunasin achieved 82.14 ± 3.34% lunasin encapsulation efficiency and improved efficacy by decreasing lunasin IC50 by 31.81% in B16-F10 and by 41.89% in A-375 compared with unencapsulated lunasin.

Rifampicin–Liposomes for Mycobacterium abscessus Infection Treatment: Intracellular Uptake and Antibacterial Activity Evaluation

Treatment of pulmonary infections caused by Mycobacterium abscessus are extremely difficult to treat, as this species is naturally resistant to many common antibiotics. Liposomes are vesicular nanocarriers suitable for hydrophilic and lipophilic drug loading, able to deliver drugs to the target site, and successfully used in different pharmaceutical applications. Moreover, liposomes are biocompatible, biodegradable and nontoxic vesicles and nebulized liposomes are efficient in targeting antibacterial agents to macrophages. The present aim was to formulate rifampicin-loaded liposomes (RIF–Lipo) for lung delivery, in order to increase the local concentration of the antibiotic. Unilamellar liposomal vesicles composed of anionic DPPG mixed with HSPC for rifampicin delivery were designed, prepared, and characterized. Samples were prepared by using the thin-film hydration method. RIF–Lipo and unloaded liposomes were characterized in terms of size, ζ-potential, bilayer features, stability and in different biological media. Rifampicin’s entrapment efficiency and release were also evaluated. Finally, biological activity of RIF-loaded liposomes in Mycobacterium abscessus-infected macrophages was investigated. The results show that RIF-lipo induce a significantly better reduction of intracellular Mycobacterium abscessus viability than the treatment with free drug. Liposome formulation of rifampicin may represent a valuable strategy to enhance the biological activity of the drug against intracellular mycobacteria.

Effect of Preexisting Immunity to Tetanus Toxoid on the Efficacy of Tetanus Toxoid-Conjugated Heroin Vaccine in Mice

Opioid use disorder (OUD) is a serious health problem that has dramatically increased over the last decade. Although current therapies for the management of OUD can be effective, they have limitations. The complementary strategy to combat the opioid crisis is the development of a conjugate vaccine to generate high affinity antibodies in order to neutralize opioids in circulation before reaching the brain. The components of an opioid vaccine include an opioid hapten (6-AmHap) that is conjugated to a carrier protein (tetanus toxoid) with the addition of adjuvants (Army Liposome Formulation adsorbed to aluminum hydroxide-ALFA). There is no consensus in the literature as to whether preexisting immunity to the carrier protein may impact the immunogenicity of the conjugate vaccine by inducing an enhanced or suppressed immune response to the hapten. Here, we investigated whether pre-exposure to tetanus toxoid would affect the immunogenicity and efficacy of the heroin vaccine, TT-6-AmHap. Mice were primed with diphtheria, tetanus, and acellular pertussis (DTaP) vaccine at weeks -4 and -2, then immunized with TT-6-AmHap vaccine at weeks 0, 3, and 6. Using ELISA and behavioral assays, we found that preexisting immunity to tetanus toxoid had no influence on the immunogenicity and efficacy of the TT-6-AmHap vaccine.

A spike-ferritin nanoparticle vaccine induces robust innate immune activity and drives polyfunctional SARS-CoV-2-specific T cells

Potent cellular responses to viral infections are pivotal for long-lived protection. Evidence is growing that these responses are critical in SARS-CoV-2 immunity. Assessment of a SARS-CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non-classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike-specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike (539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity.