Design, Fabrication and Characterization of Nanoliposomes Containing Snake Venom of Pseudocereaster percius

Background: Development of antivenom or antidote requires the repetition of immunization of large animals, such as horses and goats, which ultimately releases the IgG immunoglobulin produced in the serum specimen. As snake venom involves a variety of proteins and enzymes getting administered into the animal, this process can inflict significant harm to the animal, therefore choosing carriers that can deliver the least amount of venom could be a safer option for animal immunization Objective: In this research, nanoliposomes were used to encapsulate venom as a protected cargo for immunization. We used two distinct liposomal formulations to entrap the venom: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) associated with cholesterol in one formulation and dimethyldioctadecylamonium (Bromide salt) paired with cholesterol in the other. Method: Liposomal formulations prepared by solvent evaporation method and the venom was encapsulated in liposomes and evaluated for size and zeta potential. Meanwhile, encapsulation efficiency, venom release percentage, and phospholipase activity have all been analyzed. Results: The findings revealed that dimethyldioctadecylamonium (Bromide salt) combined with cholesterol had the highest encapsulation efficiency. In this formulation, the venom release rate had a steady-state profile. The lack of phospholipase activity in this formulation may be due to a bromide group in the liposomal structure that could be useful for immunization. Conclusion: Liposomal formulations, which do not have the active site of the snake venom enzymes, could be used for venom encapsulation.

Antibacterial Efficacy of Liposomal Formulations Containing Tobramycin and N-Acetylcysteine against Tobramycin-Resistant Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

The antibacterial activity and biofilm reduction capability of liposome formulations encapsulating tobramycin (TL), and Tobramycin-N-acetylcysteine (TNL) were tested against tobramycin-resistant strains of E. coli, K. pneumoniae and A. baumannii in the presence of several resistant genes. All antibacterial activity were assessed against tobramycin-resistant bacterial clinical isolate strains, which were fully characterized by whole-genome sequencing (WGS). All isolates acquired one or more of AMEs genes, efflux pump genes, OMP genes, and biofilm formation genes. TL formulation inhibited the growth of EC_089 and KP_002 isolates from 64 mg/L and 1024 mg/L to 8 mg/L. TNL formulation reduced the MIC of the same isolates to 16 mg/L. TNL formulation was the only effective formulation against all A. baumannii strains compared with TL and conventional tobramycin (in the plektonic environment). Biofilm reduction was significantly observed when TL and TNL formulations were used against E. coli and K. pneumoniae strains. TNL formulation reduced biofilm formation at a low concentration of 16 mg/L compared with TL and conventional tobramycin. In conclusion, TL and TNL formulations particularly need to be tested on animal models, where they may pave the way to considering drug delivery for the treatment of serious infectious diseases.

Preparation of Liposomal Formulations for Ocular Delivery of Thymoquinone: In Vitro Evaluation in HCEC-2 e HConEC Cells

Thymoquinone (TQ) is the main constituent of Nigella sativa L. essential oil. In vitro studies have shown its protective effect against H2O2-induced oxidative stress in human retinal pigment epithelium cells, and in vivo experiments have demonstrated its effect in decreasing corneal neovascularization and reducing the inflammation in an experimental dry eye model in mice. Its therapeutic use is limited by poor bioavailability, low solubility, and scarce permeability. In this study, two liposomal formulations have been developed, both of which consist of phosphatidylcholine and Plurol Oleique, a liquid lipid, and one of which is coated with 0.1% w/v hyaluronic acid (HA) to increase both TQ solubility and its ocular therapeutic potential. Each formulation has a size <200 nm and an EE% around 70%, determined by scattering techniques and the HPLC-DAD analytical method, respectively, and they result in a 2-fold increase in TQ solubility. HA-coated liposomes are stable over 2 months at +4 °C, and coated and uncoated liposomes present a gradual and prolonged release of TQ. Two cell lines, human corneal epithelial cells (HCEC-2) and human conjunctival epithelial cells (HConEC) were used to investigate the safety of the liposomal formulations. Uptake studies were also performed using fluorescent liposomes. Both liposomes and, in particular, HA-coated liposomes reduce the TQ toxicity observed at high doses in both HCEC-2 and HConEC cells, and both formulations increase the absorption at the cellular level and especially at the nucleus level, with a more pronounced effect for HA-coated liposomes.

Purification of Drug Loaded Liposomal Formulations by a Novel Stirred Cell Ultrafiltration Technique

Background: Presently reported methods for purification of liposomal formulations at laboratory scale have drawbacks of adversely affecting critical quality attributes (CQAs) of liposomes such as particle size, PDI, drug entrapment efficiency, etc., and are also not amenable for large scale processing. Objective: The present study was aimed to explore stirred cell ultrafiltration technique as a novel liposome purification method for removal of unentrapped free drug and excess external aqueous fluid, maintaining the physical integrity of liposomes. Method: Purification of brimonidine loaded liposomes (model formulation) was performed by stirred cell ultrafiltration method, and its functional performance and impact on liposomal particle size, PDI, and entrapment efficiency were compared with two widely used laboratory scale methods, i.e., ultracentrifugation and centrifugal ultrafiltration. Results: The novel stirred cell ultrafiltration method demonstrated liposomal purification within ~30 min with complete liposomal recovery showing minimal processing impact, i.e., ˂0.25 fold rise in particle size, ~0.5 fold rise in PDI, and ~4% loss in % entrapment efficiency, respectively. Whereas ultracentrifugation and centrifugal ultrafiltration methods resulted in ~4 fold and ˃2 fold rise in particle size, ˃10 fold and ˃5 fold rise in PDI, and ˃25% and ~6% loss in entrapment efficiency, respectively. Conclusion: The unique and product-friendly operational features of stirred cell ultrafiltration method demonstrated simple, rapid, and efficient liposomal purification without affecting CQAs of liposomal vesicles. This method was also evidently found to be product-friendly, rugged, versatile, and scalable up to large production batch processing, overcoming major drawbacks of presently used methods.

CD73 Downregulation by EGFR-Targeted Liposomal CD73 siRNA Potentiates Antitumor Effect of Liposomal Doxorubicin (Doxil) in 4T1 Tumor-Bearing Mice

Background Blocking CD73 ectonucleotidase has been proposed as a potential therapeutic approach for cancer treatment. The purpose of the present study was to investigate the antitumor effect of a novel EGFR-Targeted liposomal CD73 siRNA formulation in combination therapy with Doxil in the 4T1 mouse model. Methods CD73 siRNA was encapsulated into nanoliposomes by the ethanol injection method. After preparation, characterization, morphology, and stability evaluation of formulations, the toxicity was measured by MTT assay. Uptake assay and efficiency of the liposomal formulations were investigated on the 4T1 cell line. The liposomal formulation containing CD73 siRNA was targeted with GE11 peptide for in vivo evaluations. Antitumor activity of prepared formulations in combination with Doxil was studied in mice bearing 4T1 metastatic breast cancer cells. Finally, the antitumor efficacy of the formulation in concomitant treatment with Doxil was evaluated in a mouse model of breast cancer. Results The size of prepared liposomal formulations at N/P=16 for the liposomal CD73 siRNA and GE11-lipo CD73 siRNA groups were 89 nm ± 4.4 and 95 nm ± 6.6, respectively. The nanoparticle’s PDI was less than 0.3 and their surface charge was below 10 mV. The results demonstrated that N/P=16 yielded the best encapsulation efficiency which was 94% ± 3. 3. AFM results showed that the liposomes were spherical in shape and were less than 100 nm in size. The results of the MTT assay showed significant toxicity of the liposomes containing CD73 siRNA during the 48-hour cell culture. Real-time PCR and flow cytometry results showed that liposomes containing CD73 siRNA could effectively downregulate CD73 expression. Liposomal formulations were able to significantly downregulate CD73 gene expression, in vivo. However, CD73 downregulation efficiency was significantly higher for targeted form in comparison with non-targeted formulation (P-value <0.01). The combination showed maximum tumor growth delay with remarkable survival improvement compared to the control group. Studying the immune responses in the treatment groups which received doxorubicin, showed decreased number of lymphocytes in the tumor environment. However, this decrease was lower in the combination therapy group. Finally, our results clearly showed that CD73 downregulation increases the activity of CD8+ lymphocytes (INF-ℽ production) and also significantly decreases the Foxp3 in the CD25+ lymphocytes compared to the control group. Conclusion GE11-Lipo CD73 siRNA formulation can efficiently knock down CD73 ectonucleotidase. Also, the efficacy of Doxil is significantly enhanced via the downregulation of CD73 ectonucleotidase.