Leveraging immunoliposomes as nanocarriers against SARS-CoV-2 and its emerging variants

The global COVID-19 pandemic arising from SARS-CoV-2 has impacted many lives,gaining interest worldwide ever since it was first identified in December 2019.Till 2023,752 million cumulative cases and 6.8 million deaths were documented globally.COVID-19 has been rapidly evolving,affecting virus transmissibility and properties and contributing to increased disease severity.The Omicron is themost circulating variant of concern.Although success in its treatment has indicated progress in tackling the virus,limitations in delivering the current antiviral agents in battling emerging variants remain remarkable.With the latest advancements in nanotechnology for controlling infectious diseases,liposomes have the potential to counteract SARS-CoV-2 because of their ability to employ different targeting strategies,incorporating monoclonal antibodies for the active and passive targeting of infected patients.This review will present a concise summary of the possible strategies for utilizing immunoliposomes to improve current treatment against the occurrence of SARSCoV-2 and its variants.

Targeting Therapy of Choroidal Neovascularization by Use of Polypeptide-and PEDF-loaded Immunoliposomes under Ultrasound Exposure

Pigment epithelium derived factor (PEDF) has been proven to be an effective drug for the treatment of choroidal neovascularization (CNV).However,the lack of ideal administration route is the biggest bottleneck preventing PEDF from wider clinical use.In this study,we developed a novel PEDF-carrying system which employed immuno-nano-liposomes (INLs) under ultrasound exposure.PEDF-loaded INLs were prepared by conjugating nanoliposomes to the peptide ATWLPPR specifically targeting the receptor-2 for vascular endothelial growth factor (VEGFR-2) and reversely encapsuling PEDF.RF/6A cells were incubated with PEDF-loaded INLs.CNV models of BN rats were injected with PEDF-loaded INLs.MTT assay was used to evaluate the cytotoxicity of the INLs on RF/6A cells.Flow cytometry was conducted to detect the apoptotic rate of cells.Laser scanning confocal microscopy was employed to observe the binding and transmitting process of PEDF-loaded INLs and to calculate the area of CNV in the rat model.The results showed that the PEDF-loaded INLs could exclusively bind to CNV but not to the normal choroidal vessels.The CNV area was significantly decreased in PEDF treatment groups in comparison with control group (P【0.05).Moreover,PEDF-loaded INLs exposed under ultrasound were more efficient in reducing the CNV area (P【0.05).It was concluded that INLs in combination with ultrasonic exposure can transmit PEDF into cytoplasma with high specificity and efficiency,which strengthens the inhibitory effects of PEDF on CNV and reduces its side effects.PEDF-loaded INLs possibly represent a new treatment paradigm for patients with ocular neovascularization.

PREPARATION OF ANTI-LEUKEMIC T-CELL IMMUNOLIPOSOMES AND THEIR TARGET SPECIFIC PROPERTIES

Leukemic ceils exist in blood, they are easy to get in touch with target sensitive drugs. Liposomes have been extensively tested as effective, non-toxic drug carriers. Immunoliposomes, which consist of liposomes and highly specific antibodies, have been shown to bind specifically to their target cells in vitro and in vivo.In the work presented in this note, we have combined an anti-human T leukemia (?)

PREPARATION AND INTERACTION WITH TARGET CELLS OF IMMUNOLIPOSOMES FOR ESOPHAGUS CANCER CELLS ECa109

Liposomes have been extensively tested as carriers of drugs and genes. During the recent years, immunoliposomes have been developed which can carry their contents effectively to the target cells and tissues. Huang et al. have achieved the coupling of fatty acid to antibody IgG using N-hydroxysuccinimide ester for target cells. Now, we show that monoclonal antibodies IgG and IgM are derived with fatty acid using N-hydroxysuccinimide ester.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel

AIM:To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoliposomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G2/M cells and induced apoptosis, but significantly decreased the percentage of cells in G2/G1 and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

Targeting of AUF1 to vascular endothelial cells as a novel anti-aging therapy

Background Inhibition of aging of vascular endothelial cells （VECs） may delay aging and prolong life. The goal of this study was to prepare anti-CD31 monoclonal antibody conjugated PEG-modified liposomes containing the AU-rich region connecting factor 1 （AUF1） gene （CD31-PILs-AUF1） and to explore the effects of targeting CD31-PILs-AUF1 to aging VECs. Methods The mean particle sizes of various PEGylated immunoliposomes （PILs） were measured using a Zetasizer Nano ZS. Gel retardation assay was used to confirm whether PILs had encapsulated the AUF1 plasmid successfully. Fluorescence microscopy and flow cytometry were used to quantify binding of CD31-PILs-AUF1 to target cells. Flow cytometry was also used to analyze the cell cycles of aging bEnd3 cells treated with CD31-PILs-AUF1. We also developed an aging mouse model by treating mice with D-galactose. Enzyme-linked immunosorbent assay （ELISA） was used to evaluate the levels of interleuldn-6 （IL-6） and tumor necrosis factor-or （TNF-ct）. The malondialdehyde （MDA） and the superoxide dismutase （SOD） levels were detected by commercial kits. Hematoxylin-eosin （HE） staining was used to determine whether treatment with CD31-PILs-AUF 1 was toxic to the mice. Results CD31-PILs-AUF 1 specifically could targeted bEnd3 VECs and increased the percentage of cells in the S and G2/M phases of aging bEnd3 cells. ELISA showed that content of the IL-6 and TNF-ct decreased in CD31-PILs-AUF1 group. The level of SOD increased, whereas MDA decreased in the CD31-PILs-AUF1 group. Additionally, CD31-PILs-AUF 1 was not toxic to the mice. Conclusion CD31-PILs-AUF 1 targets VECs and may delay their senescence.

Preparation and Evaluation of Norcantharidin-encapsulated Liposomes Modified with a Novel CD19 Monoclonal Antibody 2E8

In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.