The hydrophobicity and high potency of many therapeutic agents makes them difficult to use effectively in clinical practice. This work focuses on conjugating phospholipid tails (2T) onto podophyllotoxin (P) and its analogue (N) using a linker and characterizing the effects of their incorporation into lipid-based drug delivery vehicles for triggered ultrasound delivery. Differential Scanning Calorimetry results show that successfully synthesized lipophilic prodrugs, 2T-P (~28 % yield) and 2T-N(~26 % yield), incorporate within the lipid membranes of liposomes. As a result of this, increased stability and incorporation are observed in 2T-P and 2T-N in comparison to the parent compounds P and N. Molecular dynamic simulation results support that prodrugs remain within the lipid membrane over a relevant range of concentrations. 2T-N's (IC50: 20 nM) biological activity was retained in HeLa cells (cervical cancer), whereas 2T-P's (IC50: ~4 µM) suffered, presumably due to steric hindrance. Proof-of-concept studies using ultrasound in vitro microbubble and nanodroplet delivery vehicles establish that these prodrugs are capable of localized drug delivery. This study provides useful information about the synthesis of double tail analogues of insoluble chemotherapeutic agents to facilitate incorporation into drug delivery vehicles. The phospholipid attachment strategy presented here could be applied to other well suited drugs such as gemcitabine, commonly known for its treatment of pancreatic cancer.
Introduction: Neuroblastomas (NB) are the most common solid tumor in children and infants, and are frequently resistant to all standard therapies. Patients are accordingly vulnerable to acute and long-term systemic effects of toxic chemotherapy. These drugs target rapidly dividing cells throughout the body, leading to severe side effects. One strategy to reduce off-target toxicities is to selectively increase drug uptake in the tumor. In pilot studies, we found that microbubbles containing liposomal doxorubicin, an agent that is effective against NB, and focused ultrasound (sonoporation) increased doxorubicin uptake in NB xenografts. However, NB is typically treated with multidrug chemotherapy, raising the importance of testing additional agents. In these studies, we evaluated a novel liposomal formulation of topotecan, an agent used in NB treatment which is both effective and associated with significant systemic toxicities. In vitro studies are needed to determine the IC50 (concentration of the drug needed to reduce the number of live cells by half). A required esterase cleavage at the delivery site can inhibit liposome encapsulated drug release. Therefore, we hypothesized that liposomal topotecan (2T-T) would have a higher IC50 than free topotecan. Methods: We tested the effect of 2T-T on nine different NB cell lines:LA1-55n, LA-1-5S, LAN-5, NGP, SK-N-AS, BE2, SHEP, NBL-WN, and SH-SY5Y;five N-type (invasive), three S-type (noninvasive), seven MYCN-amplified (poor prognosis). We have tested free topotecan in 3 of these (LA-1-5S, NBL-WN, SH-SY5Y). Cells were plated in full RPMI at 80% confluence. 2T-T or topotecan (0 to 50 uM) were added to cells 24 hours later for 72 hours. Viable cells were estimated using a WST cell counting kit. 50uM empty liposomes and lysis buffer were used as controls. Experiments were performed in triplicate, with p≥0.05deemed significant (student t-test or ratio-paired t-test using PRISM). Results: 2T-T had a mean IC50 of 0.37±0.58uM(mean R2=0.9±0.07).Empty liposomes caused no cytotoxicity in any cell line. We found no difference in IC50 according to S or N type (0.50±0.65vs 0.35±0.58(p=ns)). The mean IC50 of MYCN-amplified cells was 0.47±0.63,while that of non-MYCN-amplified was 0.031±0.029(n=2), suggesting no difference in cytotoxicity based on MYCN status. 2T-T had a 2 fold lower IC50 than free topotecan (1.14±1.19vs 0.48±0.82,p=0.046), suggesting liposomes did not inhibit topotecan release. Conclusion: These findings suggest that liposomal topotecan (T2-T) has a lower IC50 than free topotecan in NB, and that MYCN amplification and phenotype do not modify 2T-T cytotoxicity. Our results suggest that liposomal encapsulation does not inhibit topotecan release, but could increase its cytotoxicity by increasing topotecan half-life. We predict that, in vivo 2T-T could reduce toxicities and side effects, warranting the investigation of 2T-T sonoporation in NB xenografts. Citation Format: Paula Viza Gomes, Stephanie Shen, Meghan Hill, Kilkee Flynn, Mendi Marquez, Liliya Frolova, Jessica J. Kandel, Michaelann Tartis, Sonia L. Hernandez. Novel liposomal topotecan formulation has a lower IC50 than the free form on neuroblastoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1729.
Therapeutic payloads remain a challenge for phospholipid-stabilized microbubbles that are currently used as contrast agents in diagnostic ultrasound imaging. High loading is difficult to achieve due to the metastable phospholipid monolayer that lacks cargo volume, requiring lengthy drug-loaded particle tethering strategies or other sophisticated methods. The purpose of this work is to demonstrate that phospholipid conjugation can anchor chemotherapeutics to the microbubble shell with minimal disruption to phospholipid packing, resulting in an ultrasound theranostic agent that requires minimal preparation prior to administration. Using a Steglich esterification reaction, several potent chemotherapeutics were conjugated to phospholipids and were subsequently incorporated into liposomes, microbubbles, and nanodroplets for biological and particle characterization. Prodrug structures were confirmed with 1H and 13C NMR. Retention of biological activity for each phospholipid prodrug was demonstrated with an MTT cell proliferation assay using liposomes. Loading and stability of nanodroplets and microbubbles were measured with UV-Vis spectroscopy. To demonstrate site-specific delivery potential, these solutions were suspended in a submersible cell culture chamber with adhered HeLa cells. A single element transducer was used to apply a radiation force and fragmentation pulse sequence. Ultrasound-exposed and non-exposed areas treated with prodrug-containing microbubbles and nanodroplets were compared, demonstrating local efficacy.
Sonopermeation using microbubbles is an emerging extracorporeal drug delivery technique for impenetrable tumors. Utilizing the sonopermeation effect while enhancing the bioavailability of clinical chemotherapeutics using nanoparticle strategies is a potentially powerful combination. Clinically available topotecan, a chemotherapeutic, has limited bioavailability and therapeutic efficacy, which is attributed to poor solubility, rapid clearance, and inactivation under physiologic conditions. To overcome this, topotecan has been conjugated with phospholipids converting it into a lipid-prodrug (2T-T) for use in lipid-based carriers, such as liposomes, for sonopermeation. In this presentation, we present the characterization results of topotecan lipid-prodrug liposomes, preliminary in vitro efficacy in several neuroblastoma cell lines, and in vivo proof-of-concept in neuroblastoma xenograft tumors. Lipid-prodrug characterization demonstrates stable, high-drug loading liposomes are attainable while maintaining cytotoxicity. Liposome formulations, analyzed using ultraviolet-visible spectrometry and dynamic light scattering, indicate peak incorporation limits are attained for 100 nm extruded 60 mol. % 2T-T liposomes with no change in size over a 2 month period. In vivo xenograft neuroblastoma studies co-injected with lipid-prodrug liposomes (40 mol% 2T-T, 10 mg/kg) and microbubbles, for sonopermeation, were performed to determine the extent of 2T-T delivery with sonoporation over liposomal delivery alone at 24 hours post-treatment.
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