Abstracts for Short Topic Presentations (STP) Session 2: Nanomedicine and Nucleic Acid Based Therapeutics
Moderator: Veeran Kadajji
Presentation 1
Optimization of Novel Ionizable Lipid Nanoparticles for Enhanced siRNA Delivery
Mittal Darji, University of Connecticut, [email protected]
Purpose
To develop an optimized novel ionizable cationic lipid (NLD) LNP with the goal of enhancing siRNA internalization and release from endosome to improve cancer therapy.
Methods
A screening LNP formulation was developed comprising of NLD:DSPC:Cholesterol:DMG-c-PEG 2000 at 50:10:38.5:1.5 molar ratio. These LNPs were manufactured by NanoGenerator Flex-M microfluidic mixing of pH 4 acetate buffer and ethanol phases containing the nucleic acid and total lipids, respectively. Different flow rate ratio (FRR), dialysis time (DT), Nitrogen (N): Phosphorous (P) ratio were employed to assess its effect on the product. A full factorial DoE with 12 formulations was incorporated to optimize the LNP composition further. They were tested for size, surface charge, and morphology using Dynamic/Electric Light Scattering and Transmission Electron Microscopy. Encapsulation efficiency (EE) was determined using ribogreen assay. Flow cytometry and qPCR were utilized to study the in vitro transfection efficiency on MDA-MB-231 GFP cells. LNP apparent pKa and cytotoxicity of these formulations were determined.
Results
A high recovery of 90±3% was achieved for the prepared LNPs consistently. As FRR of aqueous to organic phase increased from 3:1 to 5:1, a reduction in particle size and PdI was observed from 165.4 nm, 0.137 to 90.49 nm, 0.105. A significant difference in surface charge was seen before and after dialysis from 40 mV to 3 mV showcasing the ionizable property of the novel lipid. %EE was calculated to be between 89-93. Morphology of the NLD LNP changed from collapsed to uniform structure with increased FRR. DT of 3 and 14 hours showed that the LNP size decreased with time. The apparent pKa of the novel LNP was calculated to be 5.5-5.8. N:P ratio of 5 was finalized as the optimal ratio with particle size of 105 nm, PdI 0.088, surface charge of -2 mV, 92% EE. Formulations achieved 45-60% knockdown in GFP expression. DoE study further helped to optimize the LNP composition and understand the impact of different components on the product efficacy.
Conclusion
Our optimized NLD LNP formulation shows superior physicochemical properties and promising in vitro performance. These novel LNPs hold great potential for efficient nucleic acid delivery, with implications for targeted cancer therapies.
Presentation 2
Using Coarse-Grain Molecular Dynamics to Investigate DNA-Surfactant Conjugates as a Platform for Therapeutic Nucleic Acid Delivery
Patrick Corrigan, University of Connecticut, [email protected]
Purpose
Endosomal entrapment of nucleic acid (NA) therapeutics is one of the root causes of their low delivery efficiency. To overcome this obstacle, we designed the Nucleic Acid Nanocapsule (NAN), a NA delivery vehicle designed to be degraded inside the endosome. Upon degradation, the NAN releases the oligonucleotide cargo covalently attached to a single tailed surfactant. Previous studies have shown experimentally that the surfactant conjugate increases endosomal escape efficiency, indicating the surfactant aids in NA membrane translocation. We hypothesize that more hydrophobic surfactant conjugates will lead to higher delivery efficiencies, and to that end, we have designed NANs that generate DNA-surfactant conjugates (DSCs) with two or three hydrophobic tails. Our purpose is to investigate these new DSCs using coarse-grained (CG) molecular dynamics (MD).
Methods
We have implemented CG MD to model these DSCs interacting with lipid bilayers at a near atomic resolution. Our CG DSC model was constructed in the Martini 2 forcefield and was parameterized based on all-atom (AA) MD reference data. We simulated individual DSCs interacting with a mammalian mimetic model bilayer. Steered MD/Umbrella sampling was used to calculate the energetics of DSC bilayer association.
Results
Our AA reference data showed that the surfactant conjugate tends to bundle at the terminus of the DNA. The CG umbrella sampling found that the DSCs with more hydrophobic tails had more favorable interactions with the lipid bilayer, and that all DSCs had dramatically more favorable bilayer interactions than unmodified DNA. In addition, these simulations showed that unmodified DNA prefers to sit flat on the bilayer surface, while DSCs prefer to insert into the bilayer at an angle. This allows the DSC terminal base pair to insert deeper into the bilayer than the unmodified DNA.
Conclusion
We found that more hydrophobic surfactant conjugates lead to more favorable DSC-bilayer interactions, which likely facilitate more efficient translocation and delivery. The surfactant conjugate of the DSC also allows for angled insertion of the DNA into bilayer, leading to deeper penetration of the terminal base pair into the bilayer which could possibly lead to more effective bilayer disruption and subsequent translocation.
Presentation 3
Enhancing the Stability of eGFP mRNA-LNPs: THe Role of Lyoprotectant Combinations in Post-Lyophilization Preservation
Zixuan Zhen, University of Connecticut, [email protected]
Purpose
The mRNA lipid nanoparticle (LNP) vaccines for COVID-19 have emerged as a groundbreaking advancement in the fight against the global pandemic. However, ultra-low temperature storage is necessary to maintain their efficacy and stability. The lyophilization of mRNA-LNP formulations offers a promising approach to enhance their stability. Our study aims to comprehensively understand the impact of lyoprotectants on critical quality attributes—including particle size, polydispersity index (PDI), zeta potential, mRNA encapsulation efficiency, and transfection efficiency—of freeze-dried mRNA-LNPs, to achieve a stable product post-lyophilization and after storage.
Methods
mRNAs encoding enhanced green fluorescent protein (eGFP mRNA) were loaded into LNPs. Sucrose, trehalose, sucrose with trehalose at a 1:1 (w/w) ratio, and sucrose with trehalose and PVP at a 1:1:1 (w/w) ratio were added to mRNA-LNPs with weight ratios of total lipid to lyoprotectant of 1:5, 1:10, and 1:20. Particle size, polydispersity index, and zeta potential were measured before and after lyophilization. The mRNA encapsulation efficiency was determined by the RiboGreen assay. In vitro transfection of mRNA-LNPs was assessed in HEK293T cells, with eGFP expression quantified by flow cytometry. Optimal formulations were selected for a 1-month storage stability test under 4℃, 25℃, and 40℃.
Results
Formulations with sucrose 1:20, sucrose 1:10, sucrose: trehalose=1:1 1:20, and sucrose: trehalose: PVP=1:1:1 1:20 exhibited smaller particle sizes, lower PDIs, and higher mRNA encapsulation efficiency compared to other formulations. Moreover, formulations with sucrose 1:20, sucrose: trehalose=1:1 1:20, and sucrose: trehalose: PVP=1:1:1 1:20 showed similar or even higher eGFP expression levels compared to freshly prepared mRNA-LNPs. After one month of storage at different temperatures, no significant changes were observed in particle size distribution or mRNA encapsulation efficiency, indicating good stability of the selected freeze-dried formulations.
Conclusion
This study demonstrates that sucrose, sucrose: trehalose (1:1), and sucrose: trehalose: PVP (1:1:1) at a 1:20 lipid-to-lyoprotectant ratio are effective lyoprotectants for stabilizing mRNA-LNPs during freeze-drying. These formulations preserved critical quality attributes, including particle size, PDI, and mRNA encapsulation efficiency, maintaining them comparable to before lyophilization. Furthermore, the stability of these formulations remained unchanged after one month of storage at various temperatures, indicating their potential for facilitating the development of more stable mRNA-LNP therapeutics.
Presentation 4
Evaluating Nucleic Acid Nanocapsules (NANs) as Nucleic Acid Delivery Vehicles: From Cellular to Animal Models
Jenna Cannata, University of Connecticut, [email protected]
Purpose
Nanomedicine has yielded diverse nanocarriers for delivering diagnostic and therapeutic agents. Among these, nucleic acid nanocapsules (NANs) have shown promise for nucleic acid delivery, demonstrating effective transfection and gene silencing in vitro and promising effects in in vivo disease models such as allergic airway disease. NANs comprise of a chemically crosslinked, surfactant-based micelle core displaying nucleic acid sequences on their surface. Upon delivery, esterase or proteases break down the crosslinks, releasing DNA Surfactant Conjugates (DSCs), capable of disrupting membranes and leading to cellular delivery. Herein, this work describes the evaluation of the NAN’s efficacy as a nucleic acid delivery vehicle both in vitro and in vivo.
Methods
This work first evaluates the in vitro efficacy of NANs for delivering nucleic acid therapeutics by investigating multi-tailed DSCs and their ability to improve mRNA delivery and enhance endosomal membrane disruption. Efficient endosomal escape, a key challenge in gene delivery, is crucial for the successful delivery of therapeutic nucleic acids. Multi-tailed DSCs are hypothesized to facilitate this crucial step due to their chemical structure. Second, in vivo efficacy of NANs was evaluated by examining the how they distribute within the body and whether they trigger an immune response to explore their potential therapeutic applications.
Results
We show increased protein expression in vitro with multi-tailed DSCs which suggests enhanced mRNA delivery and improved endosomal membrane disruption. The preliminary in vivo findings indicate lung accumulation after intranasal administration, which could be advantageous for treating lung-specific diseases. Furthermore, the limited immune response observed in the Luminex assay is encouraging for therapeutic applications, as it minimizes the risk of adverse effects.
Conclusion
These findings significantly advance our understanding of nucleic acid therapeutic delivery and suggest the potential of NANs as effective delivery vehicles for nucleic acid therapeutics.
Moderator: Veeran Kadajji
Presentation 1
Optimization of Novel Ionizable Lipid Nanoparticles for Enhanced siRNA Delivery
Mittal Darji, University of Connecticut, [email protected]
Purpose
To develop an optimized novel ionizable cationic lipid (NLD) LNP with the goal of enhancing siRNA internalization and release from endosome to improve cancer therapy.
Methods
A screening LNP formulation was developed comprising of NLD:DSPC:Cholesterol:DMG-c-PEG 2000 at 50:10:38.5:1.5 molar ratio. These LNPs were manufactured by NanoGenerator Flex-M microfluidic mixing of pH 4 acetate buffer and ethanol phases containing the nucleic acid and total lipids, respectively. Different flow rate ratio (FRR), dialysis time (DT), Nitrogen (N): Phosphorous (P) ratio were employed to assess its effect on the product. A full factorial DoE with 12 formulations was incorporated to optimize the LNP composition further. They were tested for size, surface charge, and morphology using Dynamic/Electric Light Scattering and Transmission Electron Microscopy. Encapsulation efficiency (EE) was determined using ribogreen assay. Flow cytometry and qPCR were utilized to study the in vitro transfection efficiency on MDA-MB-231 GFP cells. LNP apparent pKa and cytotoxicity of these formulations were determined.
Results
A high recovery of 90±3% was achieved for the prepared LNPs consistently. As FRR of aqueous to organic phase increased from 3:1 to 5:1, a reduction in particle size and PdI was observed from 165.4 nm, 0.137 to 90.49 nm, 0.105. A significant difference in surface charge was seen before and after dialysis from 40 mV to 3 mV showcasing the ionizable property of the novel lipid. %EE was calculated to be between 89-93. Morphology of the NLD LNP changed from collapsed to uniform structure with increased FRR. DT of 3 and 14 hours showed that the LNP size decreased with time. The apparent pKa of the novel LNP was calculated to be 5.5-5.8. N:P ratio of 5 was finalized as the optimal ratio with particle size of 105 nm, PdI 0.088, surface charge of -2 mV, 92% EE. Formulations achieved 45-60% knockdown in GFP expression. DoE study further helped to optimize the LNP composition and understand the impact of different components on the product efficacy.
Conclusion
Our optimized NLD LNP formulation shows superior physicochemical properties and promising in vitro performance. These novel LNPs hold great potential for efficient nucleic acid delivery, with implications for targeted cancer therapies.
Presentation 2
Using Coarse-Grain Molecular Dynamics to Investigate DNA-Surfactant Conjugates as a Platform for Therapeutic Nucleic Acid Delivery
Patrick Corrigan, University of Connecticut, [email protected]
Purpose
Endosomal entrapment of nucleic acid (NA) therapeutics is one of the root causes of their low delivery efficiency. To overcome this obstacle, we designed the Nucleic Acid Nanocapsule (NAN), a NA delivery vehicle designed to be degraded inside the endosome. Upon degradation, the NAN releases the oligonucleotide cargo covalently attached to a single tailed surfactant. Previous studies have shown experimentally that the surfactant conjugate increases endosomal escape efficiency, indicating the surfactant aids in NA membrane translocation. We hypothesize that more hydrophobic surfactant conjugates will lead to higher delivery efficiencies, and to that end, we have designed NANs that generate DNA-surfactant conjugates (DSCs) with two or three hydrophobic tails. Our purpose is to investigate these new DSCs using coarse-grained (CG) molecular dynamics (MD).
Methods
We have implemented CG MD to model these DSCs interacting with lipid bilayers at a near atomic resolution. Our CG DSC model was constructed in the Martini 2 forcefield and was parameterized based on all-atom (AA) MD reference data. We simulated individual DSCs interacting with a mammalian mimetic model bilayer. Steered MD/Umbrella sampling was used to calculate the energetics of DSC bilayer association.
Results
Our AA reference data showed that the surfactant conjugate tends to bundle at the terminus of the DNA. The CG umbrella sampling found that the DSCs with more hydrophobic tails had more favorable interactions with the lipid bilayer, and that all DSCs had dramatically more favorable bilayer interactions than unmodified DNA. In addition, these simulations showed that unmodified DNA prefers to sit flat on the bilayer surface, while DSCs prefer to insert into the bilayer at an angle. This allows the DSC terminal base pair to insert deeper into the bilayer than the unmodified DNA.
Conclusion
We found that more hydrophobic surfactant conjugates lead to more favorable DSC-bilayer interactions, which likely facilitate more efficient translocation and delivery. The surfactant conjugate of the DSC also allows for angled insertion of the DNA into bilayer, leading to deeper penetration of the terminal base pair into the bilayer which could possibly lead to more effective bilayer disruption and subsequent translocation.
Presentation 3
Enhancing the Stability of eGFP mRNA-LNPs: THe Role of Lyoprotectant Combinations in Post-Lyophilization Preservation
Zixuan Zhen, University of Connecticut, [email protected]
Purpose
The mRNA lipid nanoparticle (LNP) vaccines for COVID-19 have emerged as a groundbreaking advancement in the fight against the global pandemic. However, ultra-low temperature storage is necessary to maintain their efficacy and stability. The lyophilization of mRNA-LNP formulations offers a promising approach to enhance their stability. Our study aims to comprehensively understand the impact of lyoprotectants on critical quality attributes—including particle size, polydispersity index (PDI), zeta potential, mRNA encapsulation efficiency, and transfection efficiency—of freeze-dried mRNA-LNPs, to achieve a stable product post-lyophilization and after storage.
Methods
mRNAs encoding enhanced green fluorescent protein (eGFP mRNA) were loaded into LNPs. Sucrose, trehalose, sucrose with trehalose at a 1:1 (w/w) ratio, and sucrose with trehalose and PVP at a 1:1:1 (w/w) ratio were added to mRNA-LNPs with weight ratios of total lipid to lyoprotectant of 1:5, 1:10, and 1:20. Particle size, polydispersity index, and zeta potential were measured before and after lyophilization. The mRNA encapsulation efficiency was determined by the RiboGreen assay. In vitro transfection of mRNA-LNPs was assessed in HEK293T cells, with eGFP expression quantified by flow cytometry. Optimal formulations were selected for a 1-month storage stability test under 4℃, 25℃, and 40℃.
Results
Formulations with sucrose 1:20, sucrose 1:10, sucrose: trehalose=1:1 1:20, and sucrose: trehalose: PVP=1:1:1 1:20 exhibited smaller particle sizes, lower PDIs, and higher mRNA encapsulation efficiency compared to other formulations. Moreover, formulations with sucrose 1:20, sucrose: trehalose=1:1 1:20, and sucrose: trehalose: PVP=1:1:1 1:20 showed similar or even higher eGFP expression levels compared to freshly prepared mRNA-LNPs. After one month of storage at different temperatures, no significant changes were observed in particle size distribution or mRNA encapsulation efficiency, indicating good stability of the selected freeze-dried formulations.
Conclusion
This study demonstrates that sucrose, sucrose: trehalose (1:1), and sucrose: trehalose: PVP (1:1:1) at a 1:20 lipid-to-lyoprotectant ratio are effective lyoprotectants for stabilizing mRNA-LNPs during freeze-drying. These formulations preserved critical quality attributes, including particle size, PDI, and mRNA encapsulation efficiency, maintaining them comparable to before lyophilization. Furthermore, the stability of these formulations remained unchanged after one month of storage at various temperatures, indicating their potential for facilitating the development of more stable mRNA-LNP therapeutics.
Presentation 4
Evaluating Nucleic Acid Nanocapsules (NANs) as Nucleic Acid Delivery Vehicles: From Cellular to Animal Models
Jenna Cannata, University of Connecticut, [email protected]
Purpose
Nanomedicine has yielded diverse nanocarriers for delivering diagnostic and therapeutic agents. Among these, nucleic acid nanocapsules (NANs) have shown promise for nucleic acid delivery, demonstrating effective transfection and gene silencing in vitro and promising effects in in vivo disease models such as allergic airway disease. NANs comprise of a chemically crosslinked, surfactant-based micelle core displaying nucleic acid sequences on their surface. Upon delivery, esterase or proteases break down the crosslinks, releasing DNA Surfactant Conjugates (DSCs), capable of disrupting membranes and leading to cellular delivery. Herein, this work describes the evaluation of the NAN’s efficacy as a nucleic acid delivery vehicle both in vitro and in vivo.
Methods
This work first evaluates the in vitro efficacy of NANs for delivering nucleic acid therapeutics by investigating multi-tailed DSCs and their ability to improve mRNA delivery and enhance endosomal membrane disruption. Efficient endosomal escape, a key challenge in gene delivery, is crucial for the successful delivery of therapeutic nucleic acids. Multi-tailed DSCs are hypothesized to facilitate this crucial step due to their chemical structure. Second, in vivo efficacy of NANs was evaluated by examining the how they distribute within the body and whether they trigger an immune response to explore their potential therapeutic applications.
Results
We show increased protein expression in vitro with multi-tailed DSCs which suggests enhanced mRNA delivery and improved endosomal membrane disruption. The preliminary in vivo findings indicate lung accumulation after intranasal administration, which could be advantageous for treating lung-specific diseases. Furthermore, the limited immune response observed in the Luminex assay is encouraging for therapeutic applications, as it minimizes the risk of adverse effects.
Conclusion
These findings significantly advance our understanding of nucleic acid therapeutic delivery and suggest the potential of NANs as effective delivery vehicles for nucleic acid therapeutics.
