Transferrin-modified liposomes triggered with ultrasound to treat HeLa cells

Targeted liposomes are designed to target specific receptors overexpressed on the surfaces of cancer cells. This technique ensures site-specific drug delivery to reduce undesirable side effects while enhancing the efficiency of the encapsulated therapeutics. Upon reaching the tumor site, these liposomes can be triggered to release their content in a controlled manner using ultrasound (US). In this study, drug release from pegylated calcein-loaded liposomes modified with transferrin (Tf) and triggered with US was evaluated. Low-frequency ultrasound at 20-kHz using three different power densities (6.2 mW/cm2, 9 mW/cm2 and 10 mW/cm2) was found to increase calcein release. In addition, transferrin-conjugated pegylated liposomes (Tf-PEG liposomes) were found to be more sonosensitive compared to the non-targeted (control) liposomes. Calcein uptake by HeLa cells was found to be significantly higher with the Tf-PEG liposomes compared to the non-targeted control liposomes. This uptake was further enhanced following the exposure to low-frequency ultrasound (at 35 kHz). These findings show that targeted liposomes triggered with US have promising potential as a safe and effective drug delivery platform.

In Vivo Fluorescence Imaging of Passive Inflammation Site Accumulation of Liposomes via Intravenous Administration Focused on Their Surface Charge and PEG Modification

Nanocarriers such as liposomes have been attracting attention as novel therapeutic methods for inflammatory autoimmune diseases such as rheumatoid arthritis and ulcerative colitis. The physicochemical properties of intravenously administered nanomedicines enable them to target inflamed tissues passively. However, few studies have attempted to determine the influences of nanoparticle surface characteristics on inflammation site accumulation. Here, we aimed to study the effects of polyethylene glycol (PEG) modification and surface charge on liposome ability to accumulate in inflammatory sites and be uptake by macrophages. Four different liposome samples with different PEG modification and surface charge were prepared. Liposome accumulation in the inflammation sites of arthritis and ulcerative colitis model mice was evaluated by using in vivo imaging. There was greater PEG-modified than unmodified liposome accumulation at all inflammation sites. There was greater anionic than cationic liposome accumulation at all inflammation sites. The order in which inflammation site accumulation was confirmed was PEG-anionic > PEG-cationic > anionic > cationic. PEG-anionic liposomes had ~2.5× higher fluorescence intensity than PEG-cationic liposomes, and the PEG-liposomes had ~2× higher fluorescence intensity than non-PEG liposomes. All liposomes have not accumulated at the inflammation sites in healthy mice. Furthermore, cationic liposomes were taken up to ~10× greater extent by RAW264.7 murine macrophages. Thus, PEG-cationic liposomes that have the ability to accumulate in inflammatory sites via intravenous administration and to be taken up by macrophages could be useful.

A Novel Long-circulating DOX Liposome: Formulation and Pharmacokinetics Studies

Background: Doxorubicin (DOX) is a leading chemotherapeutic in cancer treatment because of its high potency and broad spectrum. Liposomal doxorubicin (Doxil®) is the first FDA-approved PEG-liposomes of DOX for the treatment of over 600,000 cancer patients, and it can overcome doxorubicin-induced cardiomyopathy and other side effects and prolong life span. The addition of MPEG2000-DSPE could elevate the total cost of cancer treatment. Objective: We intended to prepare a novel DOX liposome that was prepared with inexpensive materials egg yolk lecithin and Kolliphor HS15, thus allowing it to be much cheaper for clinical application. Methods: DOX liposomes were prepared using the combination of thin-film dispersion ultrasonic method and ammonium sulfate gradient method and the factors that influenced formulation quality were optimized. After formulation, particle size, entrapment efficiency, drug loading, stability, and pharmacokinetics were determined. Results: DOX liposomes were near-spherical morphology with the average size of 90 nm and polydispersity index (PDI) of less than 0.30. The drug loading was up to 7.5%, and the entrapment efficiency was over 80%. The pharmacokinetic studies showed that free DOX could be easily removed and the blood concentration of free DOX group was significantly lower than that of DOX liposomes, which indicated that the novel DOX liposome had a certain sustainedrelease effect. Conclusion: In summary, DOX liposome is economical and easy-prepared with prolonged circulation time. Lay Summary: Doxorubicin (DOX) is a leading chemotherapeutic in cancer treatment because of its high potency and broad spectrum. Liposomal doxorubicin (Doxil®) is the first FDAapproved PEG-liposomes of DOX to treat over 600.000 cancer patients, overcoming doxorubicin- induced cardiomyopathy and other side effects and prolonging life span. The addition of MPEG2000-DSPE could elevate the total cost of cancer treatment. We intend to prepare a novel DOX liposome prepared with inexpensive materials egg yolk lecithin and Kolliphor HS15, thus allowing it to be much cheaper for clinical use. The novel DOX liposome is economical and easy-prepared with prolonged circulation time.

Abstract 17412: Rapid Liposome Mimicking SARS-CoV-2 to Elucidate Thrombosis in Endothelialized Microfluidic Chip

Introduction: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), poses the worst global health crisis in modern times. Thrombotic complications such as VTE, pulmonary embolism, and risk of stroke affect more than 30% of critically ill COVID-19 patients. Unfortunately, the flow dynamics and endothelial dysfunction underlying this outcome remains poorly understood. We hereby developed liposome-nanoparticles exposing functional S-Spike (Lipo-S) to simulate the cell internalization route of SARS-CoV-2. We propose to elucidate thrombosis development in a microfluidic channel lined with HAECs exposed to Lipo-S alone, or in combination with chimeric-lentiviral vectors (ch-Lenti) expressing SARS-CoV2-ORF sequences to recapitulate SARS-CoV-2 infection. Methods: Non-PEG liposomes (100 nm) with chemically attached S-Spike proteins on their surface were developed in house. Furthermore, singular regions of SARS-CoV-2-ORFs were cloned and inserted in ch-Lenti. After exposure to Lipo-S and/or ch-Lenti vectors HAECs coverage and response will be elucidated by staining of VE-Cadherin and confocal imaging. To mimic the progression of thrombosis, whole human blood containing platelets stained with CD41-PE antibody and fluorescently labeled fibrin will be flowed with an arterial shear rate of 25 dyne/cm 2 through the microfluidic chip for 15 minutes. The chip will be fixed and imaged to characterize the clot. Results: Lipo-S showed stronger interaction for ACE2 protein compared to non-coated liposomes measured by ELISA. Our previous work showed feasibility of measuring platelet and fibrin by quantifying coverage and projecting maximum intensity of the image stack for each component. Conclusion: This novel platform will uncover mechanisms underlying COVID19 infection-mediated thrombosis, lending itself for testing therapeutics targeting selected targets with the advantage of capturing the cellular response on a rapid and small scale.

EVALUATION OF ANTI-CANCER ACTIVITY OF SURVIVIN siRNA DELIVERED BY FOLATE RECEPTOR-TARGETED POLYETHYLENE-GLYCOL LIPOSOMES IN K562-BEARING XENOGRAFT MICE

Objective: To investigate anti-cancer activity of the novel survivin siRNA Folate Receptor (FR)-targeted Polyethylene-Glycol Liposomes (PEG) liposomes in K562-bearing nude mice. Methods: The leukemia cell line K562 xenograft model was established in balb/c nu/nu mice, and survivin siRNA FR-targeted PEG liposomes was administrated by intraperitoneal (i.p.). The same volume of liposomes was administrated as placebo control. The real time PCR and western blotting were used to examine the knocking down effect. The tumor weight and size in nude mice was measured to evaluate the inhibitory effect in vivo. Results: The expression ratio of survivin mRNA in siRNA group was 0.35 ± 0.1 (survivin/GAPDH) vs. 1.85 ± 0.65 in control group with significant difference (p < 0.05), as well as protein level by western blotting analysis (p < 0.05). The results also showed the novel survivin siRNA liposomes could inhibit the growth of K562 in xenograft model. Conclusion: This novel survivin siRNA FR-targeted PEG liposome delivery system may be a potential gene therapy for the treatment of leukemia.